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  • 85
    Thermo Fisher coup tfii sirna
    Nrp2 is preferentially expressed in the AEP and the CGE and is regulated by <t>COUP-TFII.</t> ( A ) Immunohistochemical staining for Nrp2-tauGFP (green), COUP-TFII (magenta) and Lhx6 (blue) in an E13.5 horizontal section of a heterozygous Nrp2 -Δ mouse
    Coup Tfii Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/coup tfii sirna/product/Thermo Fisher
    Average 85 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    coup tfii sirna - by Bioz Stars, 2019-05
    85/100 stars
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    99
    Thermo Fisher silencer pre designed sirna
    RPLP protein downregulation induces autophagy in breast and ovarian cancer cell lines. ( A ) Numbers of MCF-7, MDA-MB-231, and OV-90 cells after transfection with RPLP0 <t>shRNA,</t> RPLP1 shRNA, <t>RPLP2</t> shRNA, or NT shRNA vector at the 3 rd d of selection with puromycin.
    Silencer Pre Designed Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/silencer pre designed sirna/product/Thermo Fisher
    Average 99 stars, based on 2438 article reviews
    Price from $9.99 to $1999.99
    silencer pre designed sirna - by Bioz Stars, 2019-05
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    91
    Santa Cruz Biotechnology p65 sirna
    The effect of MG132 protease inhibitor and <t>p65</t> <t>siRNA</t> on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p
    P65 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p65 sirna/product/Santa Cruz Biotechnology
    Average 91 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    p65 sirna - by Bioz Stars, 2019-05
    91/100 stars
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    87
    OriGene shrna sequence targeting mt1 mmp
    <t>MT1-MMP</t> overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm
    Shrna Sequence Targeting Mt1 Mmp, supplied by OriGene, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrna sequence targeting mt1 mmp/product/OriGene
    Average 87 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    shrna sequence targeting mt1 mmp - by Bioz Stars, 2019-05
    87/100 stars
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    81
    GenePharma Company anti survivin sirna
    Synthesis of NDCONH(CH 2 ) 2 <t>NH-VDGR/survivin-siRNA.</t> Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.
    Anti Survivin Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti survivin sirna/product/GenePharma Company
    Average 81 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    anti survivin sirna - by Bioz Stars, 2019-05
    81/100 stars
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    80
    GenePharma Company fluorescein labeled vegf sirna
    <t>VEGF</t> protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; <t>siRNA,</t> small interfering RNA; VEGF, vascular endothelial growth factor.
    Fluorescein Labeled Vegf Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labeled vegf sirna/product/GenePharma Company
    Average 80 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    fluorescein labeled vegf sirna - by Bioz Stars, 2019-05
    80/100 stars
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    79
    Santa Cruz Biotechnology specific sirna against pdgf rα
    Elevated TGF ‐β levels causally relate with reduced <t>PDGF</t> <t>‐Rα</t> expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P
    Specific Sirna Against Pdgf Rα, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/specific sirna against pdgf rα/product/Santa Cruz Biotechnology
    Average 79 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    specific sirna against pdgf rα - by Bioz Stars, 2019-05
    79/100 stars
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    78
    GenePharma Company fluorescein labeled survivin sirna
    Synthesis of NDCONH(CH 2 ) 2 <t>NH-VDGR/survivin-siRNA.</t> Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.
    Fluorescein Labeled Survivin Sirna, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labeled survivin sirna/product/GenePharma Company
    Average 78 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    77
    Thermo Fisher tet1 sirna
    Nanog overexpression or suppression of MAPK/ERK signaling rescues <t>Tet1</t> KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control <t>siRNA</t> and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.
    Tet1 Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tet1 sirna/product/Thermo Fisher
    Average 77 stars, based on 1 article reviews
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    tet1 sirna - by Bioz Stars, 2019-05
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    Image Search Results


    Nrp2 is preferentially expressed in the AEP and the CGE and is regulated by COUP-TFII. ( A ) Immunohistochemical staining for Nrp2-tauGFP (green), COUP-TFII (magenta) and Lhx6 (blue) in an E13.5 horizontal section of a heterozygous Nrp2 -Δ mouse

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Nrp2 is preferentially expressed in the AEP and the CGE and is regulated by COUP-TFII. ( A ) Immunohistochemical staining for Nrp2-tauGFP (green), COUP-TFII (magenta) and Lhx6 (blue) in an E13.5 horizontal section of a heterozygous Nrp2 -Δ mouse

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Immunohistochemistry, Staining

    MGE and CGE markers are coexpressed in the POa, but not in the GE. ( A and B ) Immunohistochemical staining for COUP-TFII (green) and Nkx2.1 (magenta) in an E13.5 horizontal section at the ventral ( A ) and dorsal level ( B ). Higher magnification views of

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: MGE and CGE markers are coexpressed in the POa, but not in the GE. ( A and B ) Immunohistochemical staining for COUP-TFII (green) and Nkx2.1 (magenta) in an E13.5 horizontal section at the ventral ( A ) and dorsal level ( B ). Higher magnification views of

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Immunohistochemistry, Staining

    Expression levels of COUP-TFII and Nrp2 affect the distribution of POa-derived cells into the medial amygdala. ( A ) Cell distribution of the POa-derived cells electroporated at E11.5 was examined in coronal sections of the medial amygdala at E13.5. Immunohistochemistry

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Expression levels of COUP-TFII and Nrp2 affect the distribution of POa-derived cells into the medial amygdala. ( A ) Cell distribution of the POa-derived cells electroporated at E11.5 was examined in coronal sections of the medial amygdala at E13.5. Immunohistochemistry

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Expressing, Derivative Assay, Immunohistochemistry

    Characterization of shRNA knockdown efficiency in Neuro2a cells. ( A and B ) Western blot analyses of COUP-TFII ( A ) and Nrp2 ( B ). COUP-TFII shRNA and Nrp2 shRNA efficiently deplete COUP-TFII and Nrp2 protein expression, respectively. The shRNA vectors did

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Characterization of shRNA knockdown efficiency in Neuro2a cells. ( A and B ) Western blot analyses of COUP-TFII ( A ) and Nrp2 ( B ). COUP-TFII shRNA and Nrp2 shRNA efficiently deplete COUP-TFII and Nrp2 protein expression, respectively. The shRNA vectors did

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: shRNA, Western Blot, Expressing

    Detachment of POa-derived cells from the CMS and their lateral migration following overexpression or knockdown of Nrp2 is similar to that observed following overexpression or knockdown of COUP-TFII. A coronal section series of E13.5 brains following Nrp2

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Detachment of POa-derived cells from the CMS and their lateral migration following overexpression or knockdown of Nrp2 is similar to that observed following overexpression or knockdown of COUP-TFII. A coronal section series of E13.5 brains following Nrp2

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Derivative Assay, Migration, Over Expression

    COUP-TFII overexpression does not affect the neuronal fate of the POa-derived cells. Shown is the immunohistochemical staining for DCX, GFAP, and PDGFRα in the E15.5 caudal cortex to label immature neurons, astroglia, and oligodendrocyte precursors,

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: COUP-TFII overexpression does not affect the neuronal fate of the POa-derived cells. Shown is the immunohistochemical staining for DCX, GFAP, and PDGFRα in the E15.5 caudal cortex to label immature neurons, astroglia, and oligodendrocyte precursors,

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Over Expression, Derivative Assay, Immunohistochemistry, Staining

    Migration analysis of Nrp2 knockout mice. Immunohistochemical staining for Nrp2-tauGFP (green), calbindin (red), and COUP-TFII (blue) in an E13.5 horizontal section of a heterozygous ( +/− ) or homozygous ( −/− ) Nrp2-Δ mouse

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Migration analysis of Nrp2 knockout mice. Immunohistochemical staining for Nrp2-tauGFP (green), calbindin (red), and COUP-TFII (blue) in an E13.5 horizontal section of a heterozygous ( +/− ) or homozygous ( −/− ) Nrp2-Δ mouse

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Migration, Knock-Out, Mouse Assay, Immunohistochemistry, Staining

    A population of the caudally migrating cells and the POa share molecular properties with the MGE and the CGE. ( A ) Immunohistochemical staining for COUP-TFII (green) and Lhx6 (magenta) in an E13.5 horizontal section. Multiple images were tiled and combined

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: A population of the caudally migrating cells and the POa share molecular properties with the MGE and the CGE. ( A ) Immunohistochemical staining for COUP-TFII (green) and Lhx6 (magenta) in an E13.5 horizontal section. Multiple images were tiled and combined

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Immunohistochemistry, Staining

    Nrp2 regulates detachment of migrating POa-derived cells from the CMS, downstream of COUP-TFII. ( A ) Nrp2 overexpression, Nrp2 knockdown, or rescue of COUP-TFII knockdown by Nrp2 overexpression into the POa, using in utero electroporation at E11.5, followed

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Nrp2 regulates detachment of migrating POa-derived cells from the CMS, downstream of COUP-TFII. ( A ) Nrp2 overexpression, Nrp2 knockdown, or rescue of COUP-TFII knockdown by Nrp2 overexpression into the POa, using in utero electroporation at E11.5, followed

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Derivative Assay, Over Expression, In Utero, Electroporation

    Down-regulation of COUP-TFII expression is required for POa-derived cells to distribute in the dorsal cortex. ( A and B ) Immunohistochemical staining for COUP-TFII (green) and Lhx6 (blue) in an E15.5 brain electroporated at E11.5. ( A ) Whole image of the

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Down-regulation of COUP-TFII expression is required for POa-derived cells to distribute in the dorsal cortex. ( A and B ) Immunohistochemical staining for COUP-TFII (green) and Lhx6 (blue) in an E15.5 brain electroporated at E11.5. ( A ) Whole image of the

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Expressing, Derivative Assay, Immunohistochemistry, Staining

    COUP-TFII inhibits detachment of POa-derived cells from the CMS. ( A ) Representative micrographs of an E13.5 brain electroporated into the POa at E11.5 with CAG-COUP-TFII ( Upper ), shRNA-control ( Middle ), or shRNA-COUP-TFII ( Bottom ). Median views and higher

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: COUP-TFII inhibits detachment of POa-derived cells from the CMS. ( A ) Representative micrographs of an E13.5 brain electroporated into the POa at E11.5 with CAG-COUP-TFII ( Upper ), shRNA-control ( Middle ), or shRNA-COUP-TFII ( Bottom ). Median views and higher

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Derivative Assay, shRNA

    Migration defects caused by knockdown of COUP-TFII or Nrp2 in POa-derived cells are rescued by shRNA-resistant COUP-TFII or Nrp2 expression, respectively. ( A and B ) Representative micrographs of E13.5 brains electroporated with CAG-tdTomato together with

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: Migration defects caused by knockdown of COUP-TFII or Nrp2 in POa-derived cells are rescued by shRNA-resistant COUP-TFII or Nrp2 expression, respectively. ( A and B ) Representative micrographs of E13.5 brains electroporated with CAG-tdTomato together with

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Migration, Derivative Assay, shRNA, Expressing

    The majority of caudally migrating POa-derived cells express COUP-TFII in the CMS. ( A ) Immunostaining of the cultured hemisphere electroporated into the POa2 at E13.5 for COUP-TFII (green) and Lhx6 (blue). Low magnification of the micrograph of the AEP

    Journal:

    Article Title: The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain

    doi: 10.1073/pnas.1420701112

    Figure Lengend Snippet: The majority of caudally migrating POa-derived cells express COUP-TFII in the CMS. ( A ) Immunostaining of the cultured hemisphere electroporated into the POa2 at E13.5 for COUP-TFII (green) and Lhx6 (blue). Low magnification of the micrograph of the AEP

    Article Snippet: Primary CGE cells were transfected with fluorescent protein and control or COUP-TFII siRNA (Dharmacon, SMARTpool reagent ON-TARGET plus ) using the same protocol with the MGE cells.

    Techniques: Derivative Assay, Immunostaining, Cell Culture

    RPLP protein downregulation induces autophagy in breast and ovarian cancer cell lines. ( A ) Numbers of MCF-7, MDA-MB-231, and OV-90 cells after transfection with RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector at the 3 rd d of selection with puromycin.

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: RPLP protein downregulation induces autophagy in breast and ovarian cancer cell lines. ( A ) Numbers of MCF-7, MDA-MB-231, and OV-90 cells after transfection with RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector at the 3 rd d of selection with puromycin.

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Multiple Displacement Amplification, Transfection, shRNA, Plasmid Preparation, Selection

    Upregulation of proteins related to the UPR and autophagy. MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vectors, untreated (DMSO) ( A ) or treated ( B ) with the autophagy inhibitor 3-MA (10 mM), were immunoblotted for

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: Upregulation of proteins related to the UPR and autophagy. MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vectors, untreated (DMSO) ( A ) or treated ( B ) with the autophagy inhibitor 3-MA (10 mM), were immunoblotted for

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Expressing, shRNA

    Inhibition of autophagy in cells with downregulated RPLP protein expression induces apoptosis. ( A ) Representative immunoblot of LC3, PARP1 cleavage, and proCASP6 in MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector, in the

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: Inhibition of autophagy in cells with downregulated RPLP protein expression induces apoptosis. ( A ) Representative immunoblot of LC3, PARP1 cleavage, and proCASP6 in MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector, in the

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Inhibition, Expressing, shRNA, Plasmid Preparation

    Mitochondrial depolarization and redox state perturbed in MCF-7 cells stably expressing RPLP0 shRNA, RPLP1 shRNA, or RPLP2 shRNA vectors. ( A ) RPLP0 shRNA-, RPLP1 shRNA-, and RPLP2 shRNA-associated alterations in the levels of proteins that are upregulated

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: Mitochondrial depolarization and redox state perturbed in MCF-7 cells stably expressing RPLP0 shRNA, RPLP1 shRNA, or RPLP2 shRNA vectors. ( A ) RPLP0 shRNA-, RPLP1 shRNA-, and RPLP2 shRNA-associated alterations in the levels of proteins that are upregulated

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Stable Transfection, Expressing, shRNA

    RPLP protein downregulation induces cell growth arrest. ( A ) Growth curves of MCF-7 cells stably expressing a control non-target shRNA vector (NT shRNA), or shRNA vectors targeting the RPLP0, RPLP1, or RPLP2 genes ( RPLP0 shRNA, RPLP1 shRNA, or RPLP2 shRNA,

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: RPLP protein downregulation induces cell growth arrest. ( A ) Growth curves of MCF-7 cells stably expressing a control non-target shRNA vector (NT shRNA), or shRNA vectors targeting the RPLP0, RPLP1, or RPLP2 genes ( RPLP0 shRNA, RPLP1 shRNA, or RPLP2 shRNA,

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation

    ROS-mediated autophagy in MCF-7 cells with downregulated RPLP proteins. ( A ) TEM analysis of MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector, untreated or treated with 20 mM NAC, 10 µM U0126, or

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: ROS-mediated autophagy in MCF-7 cells with downregulated RPLP proteins. ( A ) TEM analysis of MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector, untreated or treated with 20 mM NAC, 10 µM U0126, or

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Transmission Electron Microscopy, Expressing, shRNA, Plasmid Preparation

    Downregulation of proteins related to the UPR in NAC-treated MCF-7 cells with downregulated RPLP proteins. Western blot analysis of the indicated UPR-related proteins in MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector,

    Journal:

    Article Title: Disruption of the ribosomal P complex leads to stress-induced autophagy

    doi: 10.1080/15548627.2015.1063764

    Figure Lengend Snippet: Downregulation of proteins related to the UPR in NAC-treated MCF-7 cells with downregulated RPLP proteins. Western blot analysis of the indicated UPR-related proteins in MCF-7 cells expressing RPLP0 shRNA, RPLP1 shRNA, RPLP2 shRNA, or NT shRNA vector,

    Article Snippet: The following Ambion Silencer predesigned small interfering RNAs (siRNAs) (Ambion, AM16708A) were used: RPLP0 siRNA1 CCCUGAAGUG CUUGAUAUC and RPLP0 siRNA2 CGGGUACAAA CGAGUCCUGtt for the specific targeting of RPLP0 mRNA; RPLP1 siRNA1 GGAGAAGAAA GUGGA-AGCA, RPLP1 siRNA2 GGAGUCUGAU GAUGACAUGtt, and RPLP1 siRNA3 GGAGUCUGAA GAUGACAUGtt for RPLP1 ; and RPLP2 siRNA1 GGAGGAGUCU GAAGAGUCA, RPLP2 siRNA2 GGUUAUCAGU GAGCUGAAUtt, and RPLP2 siRNA3 GGAGUCUGAA GAGUCAGAUtt for RPLP2 mRNA, ATG7 siRNA1 CGCUUAACAU UGGAGUUCAG and ATG7 siRNA2 GUGUUUAUGA ACUGCCAGGU (Ambion, AM16708), and EIF2AK3 siRNA CAACAAGAAU AUCCGCAAAtt (Ambion, AM4390824).

    Techniques: Western Blot, Expressing, shRNA, Plasmid Preparation

    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on primate lentivirus infection and MX2 sensitivity. Infectivity of (A) HIV-2 or (B) SIVmac GFP reporter virus in HeLa cells stably transduced with doxycycline-inducible MX2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP214 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP214 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection and MX2 sensitivity in the presence of CsA. ( A–B ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). ( C–D ) Infectivity of HIV-1 GFP reporter virus in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HOS cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HOS cells 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Transfection, Generated, Infection

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 CA mutant infection and MX2 sensitivity. Infectivity of HIV-1 CA mutant GFP reporter viruses in HOS cells or HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2(N25)-GFP-LacZ following transfection with Nup/NTR targeting siRNA continued. HeLa cells stably transduced with MX2(N25)-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in HT1080 cells. Western blot analysis of Nup and NTR expression levels in A) dividing and B) non-dividing (aphidicolin treated) HT1080 cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. (n/a – not tested since HT1080 cells do not express NUP210, see Figure 2—figure supplement 1 ). Bands for each blot correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although blotting experiments carried out in triplicate (approximately 5), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in non-dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot is generated from an experiment in which the effect of siRNA knockdown on WT HIV-1 infection was similar to that shown in . The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HOS cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HOS cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye DRAQ5. For each plot, 3000–12000 events were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HeLa cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HeLa cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of SV40 NLS-GFP-LacZ following transfection with Nup/NTR targeting siRNA. HeLa cells stably transduced with SV40 NLS-GFP-LacZ fusion protein (green) and doxycycline-inducible MX2-RFP in the absence of doxycycline fixed and stained with Hoescht 64 hr after transfection with the indicated siRNA. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Stable Transfection, Transduction, Staining

    Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of ARFAPTIN2 fusion protein expression on lentivirus and HIV-1 CA mutant infection. ( A ) Infection of HeLa cells stably transduced with doxycycline-inducible arfaptin2 myc-tagged fusion proteins with various GFP reporter viruses in the presence (white bars) or absence (black bars) of doxycycline. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. ( B ) Infectivity of HIV-1 N57S mutant GFP reporter virus in HT1080 cells stably transduced with arfaptin2 fusion proteins in the presence (white bars) or absence (black bars) of doxycycline and the presence or absence of CsA. Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. (C) Infectivity of HIV-1-GFP reporter virus in dividing or non-dividing (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible arfaptin2 or MX2(N91)-arfaptin2 in the presence (open circles) or absence (filled circles) of doxycycline. Titers are mean ± sem, n = 3 technical replicates and are representative of three independent experiments. ( D ) Infectivity of HIV-1 GFP reporter virus in growth arrested (aphidicolin treated) HeLa cells stably transduced with doxycycline-inducible MX2(N91)-ARFAPTIN2 in the presence (open circles) or absence (filled circles) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Expressing, Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 infection in HOS cells. ( A ) Infectivity of HIV-1 GFP reporter virus in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ±sem, n = 3 technical replicates, representative of three independent experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Transfection

    Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Knockdown of Nups and NTRs in dividing HeLa cells. Western blot analysis of Nup and NTR expression levels in HeLa cells stably transduced with doxycycline-inducible MX2 64 hr after transfection with the indicated siRNA color coded by subcomplex as in Figure 4A . Red boxes highlight lanes for corresponding antibody-siRNA pairs. Bands for each Nup/NTR correspond to those indicated in Figure 2 . Each blot was generated from an experiment in which the effect of siRNA knockdown on the subcellular localization of MX2 and Nups, as well as on WT HIV-1 infection was similar to that shown below. The complete array of blots was conducted once, although randomly selected blotting experiments carried out in triplicate (approximately 10), as well as infectivity data indicated both consistent levels of knockdown and pleiotropic effects across experiments.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Western Blot, Expressing, Stable Transfection, Transduction, Transfection, Generated, Infection

    Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HeLa cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HOS cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HOS cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 6 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Cell-cycle profile of HeLa cells following transfection with Nup/NTR targeting siRNA. Cell-cycle profile of HeLa cells 64 hr after transfection with the indicated siRNA or following aphidicolin treatment, color coded by subcomplex as in Figure 4A . DNA content was determined by flow cytometry following staining with the DNA dye propidium iodide (PI). For each plot, 3000–12000 events per sample were collected. Representative of two independent experiments. 2N and 4N DNA content are indicated for the Control siRNA transfected sample.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Flow Cytometry, Cytometry, Staining

    Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Efficient siRNA-mediated knockdown of Nups and pleiotropic effects of Nup depletion in HT1080 cells. Heat map representing protein levels of Nups or NTRs (color coded by subcomplex as in Figure 4A ) in dividing (top) and non-dividing (bottom) HT1080 cells 64 hr after transfection with the indicated siRNA. Protein levels are expressed as ratios of Nup/NTR expression:LAMIN B1 expression, based on the blots shown in Figure 5—figure supplement 5 , normalized to control siRNA transfected cells that were assigned a value of 1.0 (black). Reduced expression is indicated in gray-white, with no detectable expression assigned a zero value (white). Red boxes highlight corresponding antibody-siRNA pairs.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing

    Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2 and NUP62 following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HeLa cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP62 (green), and Hoechst-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effects of depleting individual Nups on NPC integrity and function, and MX2 localization. Summary of the localization of Nups, MX2-RFP, CPSF6-RFP, or NLS-GFP-LacZ fusions upon siRNA transfection of HeLa or HT1080 cells shown in Figure 6 , and Figure 6—figure supplements 1 – 18 . Aberrant localization following siRNA transfection in ≥~80% of cells is indicated by an ‘x’ and normal localization is indicated by a dot.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection

    Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of MX2, NUP153, and RANBP2 in HT1080 cells following transfection with Nup/NTR targeting siRNA continued. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing MX2-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP153 (green), RANBP2 (purple), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 5 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Localization of CPSF6 and NUP98 in HT1080 cells following transfection with Nup/NTR targeting siRNA. Deconvolution microscopic images (single optical sections) of HT1080 cells expressing CPSF6-RFP (red, stably transduced with a doxycycline inducible vector), immunoflourescently stained NUP98 (green), and Hoescht-stained DNA. Cells were fixed and stained 64 hr after transfection with the indicated siRNA. Optical sections are approximately through the center of the vertical dimension on the nucleus. Scale bar = 10 μm. Representative of two independent experiments, with at least three images acquired per experiment.

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Transfection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Staining

    Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on HIV-1 A92E and N57S CA mutant infection in the presence of CsA. ( A–C ) Infectivity of HIV-1 A92E or N57S GFP reporter viruses in HeLa or HT1080 cells stably transduced with doxycycline-inducible MX2 in the absence of doxycycline and in the presence (asterisks) or absence (circles, HeLa or triangles, HT1080) of 5 μM CsA. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). X-axis legend for A-B is below graph B, x-axis legend for C-D is below graph D. ( D ) Infectivity of HIV-1 N57S GFP reporter virus in HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence of CsA and in the presence (open symbols) or absence (filled symbols) of doxycycline. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Mutagenesis, Infection, Stable Transfection, Transduction, Transfection

    Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Journal: eLife

    Article Title: Nuclear pore heterogeneity influences HIV-1 infection and the antiviral activity of MX2

    doi: 10.7554/eLife.35738

    Figure Lengend Snippet: Effect of Nup and NTR depletion on non-primate lentivirus infection and MX2 sensitivity. Infectivity of ( A ) EIAV or ( B-D ) FIV GFP reporter virus in ( A-B ) HeLa or ( C ) HT1080 cells stably transduced with doxycycline-inducible MX2 in the presence (open symbols) or absence (filled symbols) of doxycycline, or ( D ) HOS cells. Cells were infected 64 hr after transfection with siRNA (color coded by subcomplex as in Figure 4A ). Titers are mean ± sem, n = 3 technical replicates, representative of three independent experiments. n/a – not included due to insufficient knockdown (NUP37, RAE1) or not expressed (NUP210).

    Article Snippet: HOS, HeLa, or HT1080 cells were reverse transfected with 25 pmol of siRNA ( ; SMARTpool, Dharmacon) using Lipofectamine RNAiMax (Invitrogen) at a concentration of 3 × 104 cells/ml in 6- or 12-well plates.

    Techniques: Infection, Stable Transfection, Transduction, Transfection

    The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Collagen (I)-α1 (Col(I)α1) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Col(I)-α1 mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR

    The effect of MG132 protease inhibitor and p65 siRNA on Nestin gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Nestin mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Nestin gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. Nestin mRNA was assayed using qRT-PCR. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR

    The effect of MG132 protease inhibitor and p65 siRNA on Dentin Sialophosphoprotein (DSPP) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA (A and E, respectively), cytokine dose alone (B), and cytokine dose in the presence of MG132 or p65 siRNA (C and F, respectively) for 7 days for the examination of odontoblastic gene marker DSPP. The odontoblastic cell morphology was examined using scanning electron microscopy after 12 days (D) (for treatment IL-1β (0.5) + TNFα (1) + MG132 (0.5)). DSPP mRNA was assessed using RT-PCR. Data are presented as the mean + S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Dentin Sialophosphoprotein (DSPP) gene expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA (A and E, respectively), cytokine dose alone (B), and cytokine dose in the presence of MG132 or p65 siRNA (C and F, respectively) for 7 days for the examination of odontoblastic gene marker DSPP. The odontoblastic cell morphology was examined using scanning electron microscopy after 12 days (D) (for treatment IL-1β (0.5) + TNFα (1) + MG132 (0.5)). DSPP mRNA was assessed using RT-PCR. Data are presented as the mean + S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Marker, Electron Microscopy, Reverse Transcription Polymerase Chain Reaction

    The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression with low- and high-cytokine doses. Cells were exposed to various MG132 or p65 siRNA concentrations for 7 days. NF-κB mRNA (A, B) was assessed using qRT-PCR. NF-κB p65 protein (C, D) was assessed using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression with low- and high-cytokine doses. Cells were exposed to various MG132 or p65 siRNA concentrations for 7 days. NF-κB mRNA (A, B) was assessed using qRT-PCR. NF-κB p65 protein (C, D) was assessed using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The effect of MG132 protease inhibitor and p65 siRNA on Alkaline Phosphatase (ALP) protein expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. ALP protein was assayed using SensoLyte ALP Assay. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on Alkaline Phosphatase (ALP) protein expression in DPSCs with low- and high-cytokine doses. DPSCs were exposed to various MG132 or p65 siRNA concentrations (A and B, respectively) and cytokines in the presence of MG132 or p65 siRNA (C and D, respectively) for 7 days. ALP protein was assayed using SensoLyte ALP Assay. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, ALP Assay, Expressing

    The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression. Cells were exposed to various MG132 or p65 siRNA doses for 7 days. NF- κB mRNA (A, B) was assayed using qRT-PCR. NF-κB p65 protein expression (C, D) was evaluated using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicates statistical comparison with control result. Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by * for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on DPSC NF-κB gene expression and p65 protein expression. Cells were exposed to various MG132 or p65 siRNA doses for 7 days. NF- κB mRNA (A, B) was assayed using qRT-PCR. NF-κB p65 protein expression (C, D) was evaluated using the Chemiluminescent NF-κB p65 Transcription Factor ELISA assay:. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicates statistical comparison with control result. Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by * for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    The effect of MG132 protease inhibitor and p65 siRNA on collagen matrix formation in DPSCs with low- and high-cytokine doses. Bioquant analysis of collagen formation DPSC after 9 days’ treatment. DPSCs exposed to various inflammatory treatments in the presence or absence of MG132 (A and B) or p65 siRNA (C and D) were analyzed from picrosirius staining images using the Bioquant software. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Journal: PLoS ONE

    Article Title: Decreasing NF-κB Expression Enhances Odontoblastic Differentiation and Collagen Expression in Dental Pulp Stem Cells Exposed to Inflammatory Cytokines

    doi: 10.1371/journal.pone.0113334

    Figure Lengend Snippet: The effect of MG132 protease inhibitor and p65 siRNA on collagen matrix formation in DPSCs with low- and high-cytokine doses. Bioquant analysis of collagen formation DPSC after 9 days’ treatment. DPSCs exposed to various inflammatory treatments in the presence or absence of MG132 (A and B) or p65 siRNA (C and D) were analyzed from picrosirius staining images using the Bioquant software. Data are presented as the mean ± S. E. M. of triplicate measures from triplicate experiments. Symbols: Asterisks (*) indicate statistical comparison with control result; plus signs (+) indicate statistical comparison with IL-1β (0.5 ng/ml) and TNFα (1.0 ng/ml) treatment (low cytokine dose); triangle (Δ) indicates statistical comparison with IL-1β (10.0 ng/ml) and TNFα (20.0 ng/ml) treatment (high cytokine dose). Statistical comparison was made using ANOVA testing with Dunnett’s posthoc analysis. Statistical significance was represented by *, +, or Δ for p

    Article Snippet: A: Control, B: IL-1β (0.5 ng/ml) + TNFα (1ng/ml), C: IL-1β (0.5) + TNFα (1) + p65 siRNA (20pM), D: IL-1β (0.5) + TNFα (1) + p65 siRNA (50 pM), F: IL-1β (10) + TNFα (20), G: IL-1β (10) + TNFα (20) + p65 siRNA (20pM), H: IL-1β (10) + TNFα (20) + p65 siRNA (50 pM).

    Techniques: Protease Inhibitor, Staining, Software

    MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression inhibited the protrusive morphology of MDA-MB 231 breast cancer cells in 3D culture. a ( top ) MDA-MB 231 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown are representative fields of view of each cell line at day 5 and a respective inset. Red arrow shows a portion of the protrusive network MDA-MB 231 cells form in 3D culture. White arrows show MDA-MB 231 MT1-MMP cell colonies that have retained circularity after 5 days in 3D culture. Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MDA-MB 231 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× magnification and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. Red arrow shows protrusive MDA-MB 231 cells, whereas green arrows show circular colonies in MDA-MB 231 MT1-MMP cell lines that are positive for MT1-MMP protein signal. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Immunofluorescence, Confocal Microscopy

    MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP overexpression in MCF-7 cells induced loss of colony organization and was inversely correlated with a protrusive morphology in 3D culture. a ( top ) MCF-7 MT1-MMP cells were embedded in Matrigel and imaged every day for 5 days at 10× magnification. Shown is a representative field of view of each cell line at day 5, and indicated inset images which show the cell features quantified: Circular colonies ( white arrow ), disseminations around colonies ( green arrow ), or protrusions emanating from colonies ( red arrows ). Scale bars = 100 μm. ( bottom ) Five z-stacks per cell line were acquired every day for 5 days and disseminations and protrusions were quantified per colony for each cell line. b Representative 3D volume views of immunofluorescence analysis after MCF-7 MT1-MMP cells were embedded in Matrigel for 5 days. Samples were imaged using confocal microscopy at 60× and are displayed as overlays showing MT1-MMP signal ( green ), DAPI ( blue ) and Alexa633 phalloidin ( red ) channels. Scale bars = 100 μm. White arrow show circular colonies. Green arrows displays single cells that disseminated from the nearby colonies and show MT1-MMP protein. Red arrow shows an F-actin protrusion emanating from a circular colony ( c ) Single cells, F-actin disseminations, F-actin protrusions, and zsGreen protrusions were quantified from 20× magnification 3D volumes acquired after MCF-7 MT1-MMP cell lines stably expressing zsGreen were embedded in Matrigel for 5 days. The 60× magnification 3D volume of MT1-MMP C3 cells shows both F-actin ( red arrow ) and zsGreen ( blue arrow ) protrusions emerging from a colony. Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Immunofluorescence, Confocal Microscopy, Stable Transfection, Expressing

    Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low levels of MT1-MMP expression increased survivability of MCF-7 breast cancer cells to serum-free stress. a Viability of MCF-7 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®. b Viability of MCF-7 MT1-MMP cell lines and C3 cell line variants during incubation in SF media measured every 3 days for 9 days. Immunoblot analysis ( bottom ) shows PH3 protein levels collected from MCF-7 MT1-MMP cell lines and C3 cell line variants incubated in SF media for 6 days. β-actin was used as a loading control. Bar graph ( right ) shows densitometry analysis of PH3 levels. c Viability of MCF-7 MT1-MMP cell lines was measured after incubation for 6 days in SF media containing increasing concentrations of U0126, BB94, a furin inhibitor or an AKT inhibitor. Black asterisks show statistically significant differences between the initial and day 6 viability within cell lines, and also differences between the day 6 viability of MCF-7 cells compared to MT1-MMP expressing cell lines. Red asterisks indicate significant differences ( p ≤ 0.05) within cell lines between the day 6 viability in SF media compared to the viability after 6 days of incubation with the different concentrations of the inhibitors

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Incubation

    Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Overexpression of MT1-MMP in MDA-MB 231 cells negatively affected migration and viability ( a ) Immunoblot analysis (AB6004) showing pro-, active, and degradation forms of MT1-MMP in MDA-MB 231 breast cancer cells and three MDA-MB 231 cell lines expressing different levels of MT1-MMP . β-actin was used as a loading control. b Transwell migration assay of MDA-MB 231 MT1-MMP cells incubated in SF media for 12 h. c Viability of MDA-MB 231 MT1-MMP cell lines during incubation in media containing 10 % FBS ( top ) or serum free media ( bottom ) measured daily for 7 days using Celltiter96®

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Multiple Displacement Amplification, Migration, Expressing, Transwell Migration Assay, Incubation

    Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Schematic overview of MT1-MMP expression levels and associated changes in substrate degradation and cell migration in 2D culture, phenotypes in 3D culture, and tumourigenesis in vivo. Schematic representation of the findings of this study showing cell phenotypes across 2D and 3D culture platforms and in vivo. Legend describing molecular components in diagrams is shown at the top, and fold change relative to MCF-7 parental cells is in the brackets to the right of the bolded titles. MT1-MMP deficient breast cancer cells, such as MCF-7 cells, are incapable of proMMP-2 activation or ECM degradation, and show low migration and viability during serum-free incubation. These cells retain a circular morphology in 3D culture, and do not form vascularized tumours nor display high extravasation efficiency in vivo. Cells expressing high levels of MT1-MMP are capable of proMMP-2 activation and widespread ECM degradation, have increased survivability to serum-free stress, but do not demonstrate increased migration in 2D experiments. In 3D culture, these cells demonstrate a dissemination morphology and cell fragment release mediated by MT1-MMP. Despite MT1-MMP protein production and associated substrate degradation, these cells are unable to form vascularized tumours or increase their extravasation efficiency in vivo. Cells expressing low levels of MT1-MMP do not demonstrate proMMP-2 activation or widespread ECM degradation, but do show increased migratory potential, and high viability during serum-free incubation. These cells demonstrate a protrusive morphology in 3D culture, form vascularized tumours in vivo, and have significantly increased extravasation efficiency. Data figures within this study that correspond to the diagrams within this model are in red text

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Migration, In Vivo, Activation Assay, Incubation

    MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP activity is inversely correlated to the migratory potential of MCF-7 breast cancer cells ( a ) MCF-7 MT1-MMP cell lines stably expressing zsGreen were seeded on Alexa594-gelatin coated coverlips in media containing 0.1 % DMSO (control) or 10 μm BB94 and incubated in a live imaging chamber. Each sample was imaged at the same five stage positions every 10 mins for 20 h to visualize zsGreen cell movement and associated ECM degradation (Additional files 3, 4, 5 and 6). Shown are stills of the Alexa594 gelatin channel and an overlay including the zsGreen cells at time 0 and 20 h post-seeding of the control sample (BB94 not shown). Scale bars = 100 μm. b Time-lapse videos from ( a ) were analyzed using the ADAPT plugin for ImageJ and all individual cells tracked from each cell line were examined and grouped according to their migration distance from initial point of tracking. c Percentage of cells per field of view from each cell line that degraded the underlying AlexaFluour594-gelatin at five different time points

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Imaging, Migration

    Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Metastatic human 21 T breast cancer cells showed undetectable levels of MT1-MMP protein similar to MCF-7 C3 cells. Protein lysate from human 21 T breast cancer cell lines, which represent a progression series from atypical ductal hyperplasia (21PT-ADH), to ductal carcinoma in situ (21NT – DCIS), to invasive mammary carcinoma (21MT-1- IMC), were analyzed via immunoblot for MT1-MMP protein levels along with the MCF-7 MT1-MMP cell lines. The blots were probed with either AB6004 ( top ) or AB51074 ( bottom ) and shown as the normal exposure and as transformed versions to clearly show banding pattern. Asterisks indicate MT1-MMP isoforms (green – pro form, red- active form, orange – degradation forms). β-actin was used as a loading control

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: In Situ, Transformation Assay

    MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 MT1-MMP C3 cells displayed high tumorigenic potential when implanted onto the avian embryo CAM. MCF-7 MT1-MMP cell lines and MDA-MB 231 cells stably expressing zsGreen were implanted into the CAM of day 9 ex ovo chicken embryos and visualized 8 days post –implantation using a fluorescence stereoscope to analyze tumor vascularization. Displayed are representative bright field images showing the area of implantation on the embryo, and respective fluorescent images showing the zsGreen channel. The white boxes outline the insets showing vascularization of the MT1-MMP C3 and MDA-MB 231 tumours. Bar graph shows percentage of tumours that were vascularized ( N ≥ 11). Scale bars = 2 mm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Chick Chorioallantoic Membrane Assay, Multiple Displacement Amplification, Stable Transfection, Expressing, Fluorescence

    MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression does not correlate with increased migratory potential of breast cancer cells. a qPCR analysis of MT1-MMP mRNA levels from MCF-7, MDA-MB 231, and HS578t breast cancer cells. b Immunoblot (AB51074) and reverse zymography analysis comparing MT1-MMP, phospho-ERK and TIMP-2 protein levels between MCF-7, MDA-MB 231, and HS578t breast cancer cells. β-actin and total ERK1/2 were used as loading controls. c Gelatin zymography analysis of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 12 h. Lane 1 shows proMMP-2 CM activated by MCF-7 C2 cells treated with TIMP-2 CM diluted 1:100 to show proMMP-2 activation as a result of TIMP-2/MT1-MMP. d Transwell migration assay of MCF-7, MDA-MB 231, and HS578t breast cancer cells incubated in SF media for 24 h

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Zymography, Incubation, Activation Assay, Transwell Migration Assay

    Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Transient overexpression of MT1-MMP in MCF-7 cells did not result in increased migration and invasion ( a ) qPCR, immunoblot, and gelatin zymography analysis of MT1-MMP mRNA, protein levels, and proMMP-2 activation ability, respectively, of MCF-7 breast cancer cells transiently transfected with MT1-MMP compared to mock transfected cells (control). Immunoblot analysis (AB6004) showed pro-, active, and degradation forms of MT1-MMP protein in MT1-MMP transfected MCF-7 cells. β-actin was used as a loading control. Gelatin zymography analysis showed that MCF-7 cells transiently transfected with MT1-MMP were capable of activating proMMP-2 after 24 h of incubation as shown by intermediate and active forms of MMP-2. b Gelatin zymography analysis of MCF-7 cells transiently transfected with MT1-MMP and incubated for 12 h with serum-free media (SF, top gel) or MMP-2 conditioned media (CM, bottom gel). Lanes 1 and 2: Controls showing proMMP-2 CM chemically activated by APMA. Lanes 4 and 6: Recombinant TIMP-2 (rTIMP-2) was added at 100 ng/ml to enhance MT1-MMP-mediated proMMP-2 activation. c Immunoblot analysis of MT1-MMP transfected cells showing phospho-ERK1/2 levels. Total ERK1/2 was used as a loading control. d Transwell migration and invasion assays of MCF-7 cells transiently transfected with MT1-MMP. Number of migrated/invaded cells were normalized to control MCF-7 cells and expressed as a mean percentage ± SEM. (ns, p > 0.05 by student’s t -test)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Over Expression, Migration, Real-time Polymerase Chain Reaction, Zymography, Activation Assay, Transfection, Incubation, Recombinant

    MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines producing high levels of MT1-MMP protein demonstrated TIMP-2-mediated proMMP-2 activation ( a ) qPCR analysis of MT1-MMP mRNA from MCF-7 MT1-MMP cells lines that stably express different levels of MT1-MMP. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. b Immunoblot analysis (AB51074) showing pro-, active, and degradation forms of MT1-MMP protein in MCF-7 MT1-MMP cell lines. β-actin was used as a loading control. c Gelatin zymography analysis of MCF-7 MT1-MMP cell lines incubated for 6 or 12 h with either MMP-2 CM alone, or in combination with rTIMP-2 at 100 ng/ml, or rTIMP-2 and BB94 (10 μm). Cells were also incubated for 12 h in MMP-2 CM supplemented with TIMP-2 CM diluted 1:100 in SF media ( bottom ). Bar graph shows densitometry quantification of MMP-2 isoforms from representative zymography of MCF-7 MT1-MMP cell lines incubated with MMP-2 CM and TIMP-2 CM diluted 1:100

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Stable Transfection, Zymography, Incubation

    MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MT1-MMP expression was inversely correlated to the extravasation efficiency of MCF-7 breast cancer cells in vivo. a Representative 3D volume views at 20× magnification of MCF-7 MT1-MMP cells stably expressing zsGreen 24 h-post intravenous injection into the chicken embryo CAM vasculature. Shown is an overlay displaying the zsGreen cells ( green ) and CAM vasculature and underlying stromal vessels labeled using lectin-rhodamine ( red ), and the isolated zsGreen channel. Scale bars = 100 μm. Bar graph shows quantification of extravasation efficiency of MCF-7 MT1-MMP cell lines 24 h post-injection. b Orthogonal views of Z-stacks acquired using confocal microscopy at 60× of MT1-MMP C1 and C3 cells 24 h post-injection showing the top of the CAM capillary bed ( top ) and the underlying stroma ( bottom ). Extravasated MT1-MMP C1 cells display loss of cell fragments ( green arrows ) and membrane blebbing ( white arrow ), whereas MT1-MMP C3 cells extravasate to below the CAM with uniform morphology ( blue arrows ) and are capable of forming invasive protrusions in the stroma ( red arrow ). Scale bars = 100 μm

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Expressing, In Vivo, Stable Transfection, Injection, Chick Chorioallantoic Membrane Assay, Labeling, Isolation, Confocal Microscopy

    Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: Low MT1-MMP/high TIMP-2 was optimal to promote migration and ERK activation in MCF-7 cells. a ERK activation in MCF-7 and MT1-MMP cells after incubation for 12 h (top) or 15 min ( bottom ) in media containing 10 % FBS or different dilutions of TIMP-2 or ALA + TIMP-2 CM in SF media. b Scratch closure migration assay of MCF-7 MT1-MMP cell lines monitored for 3 days. Shown are representative 10× fields of view. The white dotted lines indicate the initial scratch size; red dotted lines indicate the scratch size at the respective day. Scale bars = 100 μm. Line graph on the right shows scratch closure quantification that demonstrates significantly increased migratory potential of C3 cells. c Transwell migration assays of MCF-7 MT1-MMP cell lines incubated for 48 h in TIMP-2, or ALA + TIMP-2 CM diluted 1:100 ( top ), or ALA + TIMP-2 CM in increasing dilutions ( bottom ). d ( top ) qPCR analysis showing MT1-MMP mRNA from two cell lines derived from MT1-MMP C3 cells that stably express an shRNA construct targeting MT1-MMP, and one cell line stably expressing a control scrambled shRNA construct. Different letters indicate significant differences at p ≤ 0.05 by one-way ANOVA, Tukey’s post-hoc test. Individual student’s t-tests comparing MCF-7 cells against the C3 SH 1 cell line is also shown. ( bottom ) Transwell migration assay of MT1-MMP C3 cell lines incubated for 48 h in either TIMP-2 or ALA + TIMP-2 CM diluted 1:10, or ALA + TIMP2/U0126 (10 μm)

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Migration, Activation Assay, Incubation, Real-time Polymerase Chain Reaction, Derivative Assay, Stable Transfection, shRNA, Construct, Expressing, Transwell Migration Assay

    MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Journal: Molecular Cancer

    Article Title: Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

    doi: 10.1186/s12943-016-0547-x

    Figure Lengend Snippet: MCF-7 cell lines that express high levels of MT1-MMP demonstrated widespread ECM degradation (a) MCF-7, MT1-MMP cell lines, and MDA-MB 231 breast cancer cells were incubated on Alexa488 gelatin-coated coverslips for 24 h and processed for immunofluorescence to examine cytoplasmic MT1-MMP protein and ECM degradation. Representative fields of view are shown at 20× ( top panels ) and 60× ( bottom panels ) magnification. Panels are composed of an overlay showing the nuclei, F-actin, and Alexa488 gelatin signal ( top ) and the MT1-MMP signal with an inset of the Alexa488 gelatin channel ( bottom ). White arrows indicate cells that have degraded the underlying gelatin but are devoid of MT1-MMP signal. Scale bars = 100 μm. Cells in each sample positive for cytoplasmic MT1-MMP protein signal (MT1-MMP +) or devoid of underneath Alexa488 gelatin signal (Gelatin -) were quantified per 20× fields of view and are shown as mean percentage of total cells per field of view ± SEM

    Article Snippet: Stable cells lines expressing an shRNA sequence targeting MT1-MMP in the vector pRS (TR311445, Origene) were generated in the same manner expect using puromycin (2 μg/ml) as the selection antibiotic.

    Techniques: Multiple Displacement Amplification, Incubation, Immunofluorescence

    Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Microscopy, Small Interfering RNA

    The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Electron Microscopy, Small Interfering RNA

    Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation

    Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Mass Spectrometry, Small Interfering RNA

    TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy, Small Interfering RNA

    Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    VEGF protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: VEGF protein expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, Small Interfering RNA

    Scheme for GPF and GPF/DOX/VEGF-siRNA preparation. (I) KOH, 70°C, 24 hours, DI water. (II) Room temperature, 24 hours. (III) Stirred at room temperature, without light, 12 hours, DI water. (IV) Incubation at 37°C, 30 minutes, DEPC water. Abbreviations: DEPC, diethyl pyrocarbonate; DI, deionized; DOX, doxorubicin; FA, folic acid; GO, graphene oxide; GPF, GO-PLL/FA; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Scheme for GPF and GPF/DOX/VEGF-siRNA preparation. (I) KOH, 70°C, 24 hours, DI water. (II) Room temperature, 24 hours. (III) Stirred at room temperature, without light, 12 hours, DI water. (IV) Incubation at 37°C, 30 minutes, DEPC water. Abbreviations: DEPC, diethyl pyrocarbonate; DI, deionized; DOX, doxorubicin; FA, folic acid; GO, graphene oxide; GPF, GO-PLL/FA; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Incubation, Small Interfering RNA

    Confocal images of the HeLa cells. ( A ) Blank control, ( B ) naked FAM-VEGF-siRNA, ( C ) DOX, ( D ) GPF/FAM-VEGF-siRNA, ( E ) GPF/DOX, ( F ) Lipo™2000/FAM-VEGF-siRNA, and ( G ) GPF/DOX/FAM-VEGF-siRNA. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Confocal images of the HeLa cells. ( A ) Blank control, ( B ) naked FAM-VEGF-siRNA, ( C ) DOX, ( D ) GPF/FAM-VEGF-siRNA, ( E ) GPF/DOX, ( F ) Lipo™2000/FAM-VEGF-siRNA, and ( G ) GPF/DOX/FAM-VEGF-siRNA. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Image of tumors of blank control, positive control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Image of tumors of blank control, positive control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Positive Control, Small Interfering RNA

    The tumor weights of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, DOX (positive control), and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The tumor weights of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, DOX (positive control), and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Positive Control, Small Interfering RNA

    The tumor volume of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The tumor volume of blank control, naked VEGF-siRNA, DOX, GPF/VEGF-siRNA, GPF/DOX, and GPF/DOX/VEGF-siRNA (n=10). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Degradation of GPF/VEGF-siRNA with heparin and anti-RNase A. Abbreviations: GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Degradation of GPF/VEGF-siRNA with heparin and anti-RNase A. Abbreviations: GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Agarose gel electrophoresis retardation assays of VEGF-siRNA complexed with GPF ( A ) and GO ( B ). Abbreviations: GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Agarose gel electrophoresis retardation assays of VEGF-siRNA complexed with GPF ( A ) and GO ( B ). Abbreviations: GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    VEGF mRNA expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: VEGF mRNA expression of HeLa cells treated with different medicines. Data are presented as the mean ± SD, n=3. Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, Small Interfering RNA

    Zeta potential measurements of GO ( A ), GO-PLL ( B ), GPF ( C ), and GPF/DOX/VEGF-siRNA ( D ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-PLL/folic acid; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Zeta potential measurements of GO ( A ), GO-PLL ( B ), GPF ( C ), and GPF/DOX/VEGF-siRNA ( D ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-PLL/folic acid; PLL, poly- l -lysine hydrobromide; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    Anti-proliferation effect of GPF/DOX/VEGF-siRNA on HeLa cells (n=3). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: Anti-proliferation effect of GPF/DOX/VEGF-siRNA on HeLa cells (n=3). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NC, normal control; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Small Interfering RNA

    The expression of VEGF protein in vivo (n=5). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: The expression of VEGF protein in vivo (n=5). Abbreviations: DOX, doxorubicin; GPF, graphene oxide-poly- l -lysine hydrobromide/folic acid; NS, normal saline; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Expressing, In Vivo, Small Interfering RNA

    TEM images of GO ( A ), GPF ( B ), and GPF/DOX/VEGF-siRNA ( C ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor.

    Journal: International Journal of Nanomedicine

    Article Title: Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo

    doi: 10.2147/IJN.S162939

    Figure Lengend Snippet: TEM images of GO ( A ), GPF ( B ), and GPF/DOX/VEGF-siRNA ( C ). Abbreviations: DOX, doxorubicin; GO, graphene oxide; GPF, GO-poly- l -lysine hydrobromide/folic acid; siRNA, small interfering RNA; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor.

    Article Snippet: VEGF-siRNA and fluorescein-labeled VEGF-siRNA (FAM-VEGF-siRNA, Cy3-VEGF-siRNA) were purchased from Gene-Pharma Co, Ltd (Shanghai, China).

    Techniques: Transmission Electron Microscopy, Small Interfering RNA, Transmission Assay, Electron Microscopy

    Elevated TGF ‐β levels causally relate with reduced PDGF ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Elevated TGF ‐β levels causally relate with reduced PDGF ‐Rα expression in nCLD patients and mice undergoing MV ‐O 2 , reducing downstream signaling and migration in pulmonary myofibroblasts Representative immunofluorescence images showing reduced expression of PDGF‐Rα (red) in the lungs of patients ( n = 7) developing nCLD (lower left panel, red stain; white arrows) together with increased pSMAD‐2 expression (lower middle panel, green stain; white arrows) as compared to lung sections from a non‐nCLD patient ( n = 1) (upper panel) (200×). Negative correlation between PDGF‐Rα and TGF‐β1 in transcriptome analysis 72 h after birth in preterms with nCLD ( n = 11) in contrast to non‐nCLD ( n = 9); z test of the difference of the Fisher's z transformed correlations divided by the standard error of the difference ( P = 0.048); scatter plots log2‐gene expression; linear regression (blue), with 95% CI (gray). MV‐O 2 reduced lung PDGF‐Rα (main: red stain; white arrows) and increased pSMAD‐2 levels (insert: red stain; white arrows) in ventilated neonatal WT (lower panel) when compared to control WT mice (upper panel); ( n = 4 mice/group; 10 images/mouse; 200×). Luciferase assay of CCL‐206 cells transfected with pGL4.14 containing PDGF‐Rα promoter revealing reduced promoter activity upon TGF‐β application (normalized to control) ( n = 3 experiments). Immunoblot analysis showing reduced PDGF‐Rα (E), VEGF‐A (F), and pERK/EKR (G) protein levels upon TGF‐β application alone in primary pulmonary myofibroblasts from 5–7‐day‐old WT mice ( n = 6–9 mice/group). Reduced migration of myofibroblasts (MFBs) from neonatal WT mice upon TGF‐β application alone ( n = 5 mice/group, 3 technical replicates). Translation of the results in fibroblasts isolated from tracheal aspirates of ventilated preterm infants (hMFBs) displayed reduced PDGF‐Rα levels (I) and migration assessed by Boyden chamber assay (J) upon TGF‐β application ( n = 3–5 patients/group). Representative phase contrast images (100×) of scratch migration assays in human lung fibroblasts (hMFBs) after 48 h of TGF‐β incubation indicating decreased wound closure (K) quantified by reduced velocity (L) and distance travelled (M) ( n = 3 patients/group). Data information: In (D–J) and (L, M), data are presented as mean ± SD and normalized to control. Statistical test used is two‐tailed unpaired Student's t ‐test or Mann–Whitney test ( P = 0.0002–0.039). *** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Expressing, Mouse Assay, Migration, Immunofluorescence, Staining, Transformation Assay, Luciferase, Transfection, Activity Assay, Isolation, Boyden Chamber Assay, Incubation, Two Tailed Test, MANN-WHITNEY

    Effect of TGF ‐β in combination with mechanical stretch on PDGF ‐Rα signaling and functional properties of fibroblasts No change in migration of myofibroblasts (MFBs) from newborn WT mice with mechanical stretch ( n = 5 mice/group). Analysis of proliferation (Cell Titer Glo) exhibited no change upon TGF‐β (B) or stretch (C) in mouse myofibroblasts (MFBs) from WT mice, while an increase in proliferation was observed when mouse myofibroblasts stretched in the presence of TGF‐β were subjected to an additional dose of TGF‐β (D) ( n = 9 mice/group). Immunoblot analysis showing increased proliferation markers like PCNA (E) levels with mechanical stretch and PI3K (F) upon additional TGF‐β incubation on stretched myofibroblasts (MFBs) from newborn WT mice ( n = 6 mice/group). Data information: Values here are normalized to respective controls. Data are presented as mean ± SD. Statistical analysis in (A–C, E) is two‐tailed unpaired Student's t ‐test and in (D, F) is ordinary one‐way ANOVA with Bonferroni's correction. ** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Effect of TGF ‐β in combination with mechanical stretch on PDGF ‐Rα signaling and functional properties of fibroblasts No change in migration of myofibroblasts (MFBs) from newborn WT mice with mechanical stretch ( n = 5 mice/group). Analysis of proliferation (Cell Titer Glo) exhibited no change upon TGF‐β (B) or stretch (C) in mouse myofibroblasts (MFBs) from WT mice, while an increase in proliferation was observed when mouse myofibroblasts stretched in the presence of TGF‐β were subjected to an additional dose of TGF‐β (D) ( n = 9 mice/group). Immunoblot analysis showing increased proliferation markers like PCNA (E) levels with mechanical stretch and PI3K (F) upon additional TGF‐β incubation on stretched myofibroblasts (MFBs) from newborn WT mice ( n = 6 mice/group). Data information: Values here are normalized to respective controls. Data are presented as mean ± SD. Statistical analysis in (A–C, E) is two‐tailed unpaired Student's t ‐test and in (D, F) is ordinary one‐way ANOVA with Bonferroni's correction. ** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Functional Assay, Migration, Mouse Assay, Incubation, Two Tailed Test

    Reduced PDGF ‐Rα abundance in PDGF ‐Rα haploinsufficient mice Double staining for PDGF‐Rα (red) and α‐SMA (green) showed a co‐localization of these two proteins as well as a reduced number of double‐positive cells (white arrows) in 5–8‐day‐old PDGF‐Rα +/− mice (right panel) when compared to PDGF‐Rα +/+ (WT) littermates (left panel). * represents alveolar air space. (B) Quantitative RT–PCR and (C) immunoblot analysis showing reduced PDGF‐Rα protein and mRNA expression in lungs of PDGF‐Rα +/− newborn mice when compared to WT littermates ( n = 4/group). Reduced PDGF‐Rα transcript in newborn PDGF‐Rα +/− mice upon MV‐O 2 for 8 h when compared to WT littermates ( n = 3 mice/group). Data information: In (B–D), the data are presented as mean ± SD. * P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Reduced PDGF ‐Rα abundance in PDGF ‐Rα haploinsufficient mice Double staining for PDGF‐Rα (red) and α‐SMA (green) showed a co‐localization of these two proteins as well as a reduced number of double‐positive cells (white arrows) in 5–8‐day‐old PDGF‐Rα +/− mice (right panel) when compared to PDGF‐Rα +/+ (WT) littermates (left panel). * represents alveolar air space. (B) Quantitative RT–PCR and (C) immunoblot analysis showing reduced PDGF‐Rα protein and mRNA expression in lungs of PDGF‐Rα +/− newborn mice when compared to WT littermates ( n = 4/group). Reduced PDGF‐Rα transcript in newborn PDGF‐Rα +/− mice upon MV‐O 2 for 8 h when compared to WT littermates ( n = 3 mice/group). Data information: In (B–D), the data are presented as mean ± SD. * P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Mouse Assay, Double Staining, Quantitative RT-PCR, Expressing

    Supplemental PDGF ‐A rescues both the air sac and microvascular nCLD phenotypes induced by MV ‐O 2 in neonatal PDGF ‐Rα haploinsufficient mice Improved alveolar structure in 5–8‐day‐old PDGF‐Rα +/− mice undergoing 8 h of MV‐O 2 after intra‐tracheal treatment with PDGF‐A (10 μl/g bw, 25 ng/ml PDGF‐A) when compared to mice receiving sterile saline (200×), confirmed by quantitative image analysis with increased alveolar counts (B) as well as vessel number normalized to 100 alveoli (C) (20–100 μm; n = 2–4 mice/group). Immunoblot analysis of total lung homogenates showed increased PDGF‐Rα (D) together with increased JAK‐2 (E), STAT‐3 (F), VEGF‐A (G), VE‐cadherin (H), and AKT (I) protein levels in PDGF‐A‐treated PDGF‐Rα +/− mice after 8 h of MV‐O 2 when compared to WT littermates ( n = 3–4 mice/group). Panels (D, H) and (E, F) are from same blot hence having same β‐actin bands. Quantitative image analysis indicated increased VEGF‐A to PDGF‐Rα protein levels (J, M upper panel) together with an increase in CD31 expression in relation to total tissue (K, M lower panel) and a decrease in apoptotic (cleaved caspase‐3) CD31‐expressing cells (L) in the lungs of PDGF‐A‐treated PDGF‐Rα +/− mice when compared to saline‐treated controls after 8 h of MV‐O 2 ( n = 2–4 mice/group). Upper panel in (M) shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with VEGF‐A (green), PDGF‐Rα (red) dual positive (orange, white arrows), and inserts show VEGF‐A stain (green). Lower panel shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with cleaved caspase‐3 (green), CD31 (red) dual positive (orange, white arrows). Nucleus is stained with DAPI (blue). Treatment with PDGF‐A in ventilated neonatal PDGF‐Rα +/− mice led to improved lung compliance displayed as a function of airway pressure (Ptramax) and tidal volume when compared to untreated mice ( n = 4 mice/group). Data information: In (B–L, N), the data are presented as mean ± SD. *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Supplemental PDGF ‐A rescues both the air sac and microvascular nCLD phenotypes induced by MV ‐O 2 in neonatal PDGF ‐Rα haploinsufficient mice Improved alveolar structure in 5–8‐day‐old PDGF‐Rα +/− mice undergoing 8 h of MV‐O 2 after intra‐tracheal treatment with PDGF‐A (10 μl/g bw, 25 ng/ml PDGF‐A) when compared to mice receiving sterile saline (200×), confirmed by quantitative image analysis with increased alveolar counts (B) as well as vessel number normalized to 100 alveoli (C) (20–100 μm; n = 2–4 mice/group). Immunoblot analysis of total lung homogenates showed increased PDGF‐Rα (D) together with increased JAK‐2 (E), STAT‐3 (F), VEGF‐A (G), VE‐cadherin (H), and AKT (I) protein levels in PDGF‐A‐treated PDGF‐Rα +/− mice after 8 h of MV‐O 2 when compared to WT littermates ( n = 3–4 mice/group). Panels (D, H) and (E, F) are from same blot hence having same β‐actin bands. Quantitative image analysis indicated increased VEGF‐A to PDGF‐Rα protein levels (J, M upper panel) together with an increase in CD31 expression in relation to total tissue (K, M lower panel) and a decrease in apoptotic (cleaved caspase‐3) CD31‐expressing cells (L) in the lungs of PDGF‐A‐treated PDGF‐Rα +/− mice when compared to saline‐treated controls after 8 h of MV‐O 2 ( n = 2–4 mice/group). Upper panel in (M) shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with VEGF‐A (green), PDGF‐Rα (red) dual positive (orange, white arrows), and inserts show VEGF‐A stain (green). Lower panel shows sections from PDGF‐Rα +/− mice treated with NaCl (left) or PDGF‐A (right) stained with cleaved caspase‐3 (green), CD31 (red) dual positive (orange, white arrows). Nucleus is stained with DAPI (blue). Treatment with PDGF‐A in ventilated neonatal PDGF‐Rα +/− mice led to improved lung compliance displayed as a function of airway pressure (Ptramax) and tidal volume when compared to untreated mice ( n = 4 mice/group). Data information: In (B–L, N), the data are presented as mean ± SD. *** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Mouse Assay, Expressing, Staining

    Enrichment of PDGF ‐Rα SNP s associated with reduced protein levels and migration of lung fibroblasts from ventilated preterm infants developing nCLD Decreased PDGF‐Rα expression in human lung fibroblasts (hMFBs) from preterms undergoing MV‐O 2 (21.7 ± 8 vs. 4.7 ± 1 day of life; serial samples 2 5, 3 6; n = 3 patients/group). Regional association plot showing −log10 P ‐values ( y ‐axis) of SNPs according to chromosomal positions ( x ‐axis). Light blue: estimated recombination rate (cM/Mb, HapMap CEU population); blue: most significant SNP (rs12506783); red: r 2 ≥ 0.8; orange: 0.8 > r 2 ≥ 0.5, yellow: 0.5 > r 2 ≥ 0.2, gray: 0.2 > r 2 ≥ 0. P ‐values were determined using R by case‐control analysis with a logistic regression model including case/control status, sex, gestational age at birth, status “small for gestational age”, and country of origin of the mother. Analysis was adjusted for relatedness to account for multiple births (R‐package GenABEL). Levels of PDGF‐Rα gene expression in patients ( n = 9), which are carrying at least one SNP (minor allele) compared to patients with no SNPs (major allele). Major alleles are given in the figure labels. Minor alleles in rs10022540 are A, in rs11133311 are T, and in rs12506783 are C . PDGF‐R (D) and VEGF‐A (E) protein levels in separate patient cohort ( n = 13) carrying at least one SNP (minor allele) at position rs12506783 compared to patients with no SNPs (major allele). Protein levels were quantified using SOMAlogic technique. Data are presented as mean ± SD. Two‐tailed unpaired Student's t ‐test (* P = 0.0336; # P = 0.0863). Representative PDGF‐Rα levels (F) and migratory potential assessed by Boyden chamber assay (G) in fibroblasts isolated from tracheal aspirates of patients with nCLD. The fibroblast carrying SNP at both alleles (homozygote minor allele) displayed reduced PDGF‐Rα levels and migration when compared to fibroblasts from patients carrying SNP at one allele (heterozygote minor allele). Data are presented as mean ± SD ( n = 3/4 replicates). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Enrichment of PDGF ‐Rα SNP s associated with reduced protein levels and migration of lung fibroblasts from ventilated preterm infants developing nCLD Decreased PDGF‐Rα expression in human lung fibroblasts (hMFBs) from preterms undergoing MV‐O 2 (21.7 ± 8 vs. 4.7 ± 1 day of life; serial samples 2 5, 3 6; n = 3 patients/group). Regional association plot showing −log10 P ‐values ( y ‐axis) of SNPs according to chromosomal positions ( x ‐axis). Light blue: estimated recombination rate (cM/Mb, HapMap CEU population); blue: most significant SNP (rs12506783); red: r 2 ≥ 0.8; orange: 0.8 > r 2 ≥ 0.5, yellow: 0.5 > r 2 ≥ 0.2, gray: 0.2 > r 2 ≥ 0. P ‐values were determined using R by case‐control analysis with a logistic regression model including case/control status, sex, gestational age at birth, status “small for gestational age”, and country of origin of the mother. Analysis was adjusted for relatedness to account for multiple births (R‐package GenABEL). Levels of PDGF‐Rα gene expression in patients ( n = 9), which are carrying at least one SNP (minor allele) compared to patients with no SNPs (major allele). Major alleles are given in the figure labels. Minor alleles in rs10022540 are A, in rs11133311 are T, and in rs12506783 are C . PDGF‐R (D) and VEGF‐A (E) protein levels in separate patient cohort ( n = 13) carrying at least one SNP (minor allele) at position rs12506783 compared to patients with no SNPs (major allele). Protein levels were quantified using SOMAlogic technique. Data are presented as mean ± SD. Two‐tailed unpaired Student's t ‐test (* P = 0.0336; # P = 0.0863). Representative PDGF‐Rα levels (F) and migratory potential assessed by Boyden chamber assay (G) in fibroblasts isolated from tracheal aspirates of patients with nCLD. The fibroblast carrying SNP at both alleles (homozygote minor allele) displayed reduced PDGF‐Rα levels and migration when compared to fibroblasts from patients carrying SNP at one allele (heterozygote minor allele). Data are presented as mean ± SD ( n = 3/4 replicates). Source data are available online for this figure.

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Migration, Expressing, Two Tailed Test, Boyden Chamber Assay, Isolation

    PDGF ‐Rα haploinsufficiency drives reduced pulmonary micro‐vessel density with increased endothelial cell apoptosis in neonatal mice undergoing MV ‐O 2 Histologic and immunoblot analysis displayed reduced small vessel number (20–100 μm diameter) normalized to 100 alveoli as well as reduced pulmonary VEGF‐R2, VE‐cadherin, and VEGF‐A protein levels, respectively ( n = 6–8 mice/group). Panels (B) and (C) are from same blot hence having same β‐actin bands. Immunofluorescence images of lung tissue (400×; merged) from neonatal PDGF‐Rα +/− mice indicating increased cleaved caspase‐3 (red, white arrows; lower panel) after 8 h of MV‐O 2 in contrast to WT mice (upper panel; green: CD31; blue: DAPI). Double stain revealed increased cleaved caspase‐3 + /CD31 + cells normalized to CD31 area in PDGF‐Rα +/− mice after 8 h of MV‐O 2 ( n = 4 mice/group, 4 sections/mice, and 10 images/section). Representative image confirming increased endothelial apoptosis in neonatal PDGF‐Rα +/− mice after 8 h of MV‐O 2 (lower panel; white arrows) with VE‐cadherin (red) and cleaved caspase‐3 (green) and nucleus stained with DAPI (blue) when compared to WT mice (upper panel) ( n = 2 mice/group). Increased caspase‐3 activation in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 experiments). In vitro application of PDGF‐Rα siRNA to primary lung mouse myofibroblasts from WT mice diminished PDGF‐Rα (H) protein (normalized to control), associated with reduced VEGF‐A protein (I) ( n = 3 mice/group). Increased cleaved caspase‐9 and reduced eNOS protein levels in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 mice/group). Data information: Data are presented as mean ± SD. *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: PDGF ‐Rα haploinsufficiency drives reduced pulmonary micro‐vessel density with increased endothelial cell apoptosis in neonatal mice undergoing MV ‐O 2 Histologic and immunoblot analysis displayed reduced small vessel number (20–100 μm diameter) normalized to 100 alveoli as well as reduced pulmonary VEGF‐R2, VE‐cadherin, and VEGF‐A protein levels, respectively ( n = 6–8 mice/group). Panels (B) and (C) are from same blot hence having same β‐actin bands. Immunofluorescence images of lung tissue (400×; merged) from neonatal PDGF‐Rα +/− mice indicating increased cleaved caspase‐3 (red, white arrows; lower panel) after 8 h of MV‐O 2 in contrast to WT mice (upper panel; green: CD31; blue: DAPI). Double stain revealed increased cleaved caspase‐3 + /CD31 + cells normalized to CD31 area in PDGF‐Rα +/− mice after 8 h of MV‐O 2 ( n = 4 mice/group, 4 sections/mice, and 10 images/section). Representative image confirming increased endothelial apoptosis in neonatal PDGF‐Rα +/− mice after 8 h of MV‐O 2 (lower panel; white arrows) with VE‐cadherin (red) and cleaved caspase‐3 (green) and nucleus stained with DAPI (blue) when compared to WT mice (upper panel) ( n = 2 mice/group). Increased caspase‐3 activation in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 experiments). In vitro application of PDGF‐Rα siRNA to primary lung mouse myofibroblasts from WT mice diminished PDGF‐Rα (H) protein (normalized to control), associated with reduced VEGF‐A protein (I) ( n = 3 mice/group). Increased cleaved caspase‐9 and reduced eNOS protein levels in HUVECs upon incubation with supernatants for 6 h obtained from lung mouse myofibroblasts after PDGF‐Rα siRNA treatment when compared to control siRNA ( n = 3 mice/group). Data information: Data are presented as mean ± SD. *** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Activation Assay, Incubation, In Vitro

    PDGF ‐Rα haploinsufficiency drives the air sac pathology of nCLD in neonatal mice undergoing MV ‐O 2 Representative lung tissue sections (200×) from 5–8‐day‐old PDGF‐Rα +/+ (WT) and PDGF‐Rα +/− mice after 8 h of MV‐O 2 showing increased air space size compared to respective controls (O 2 ‐control) spontaneously breathing 40% O 2 for 8 h. Quantitative analysis of lung tissue sections showed increased alveolar area after 8 h of MV‐O 2 in PDGF‐Rα +/− mice, whereas no significant change was observed in WT mice when compared to respective controls ( n = 6–11 mice/group). Radial alveolar counts (alveolar number) in lung tissue sections from WT and PDGF‐Rα +/− mice were reduced after 8 h of MV‐O 2 when compared to respective controls ( n = 6–11 mice/group). Septal density was significantly reduced in PDGF‐Rα +/− mice when compared to WT littermates after 8 h of MVO 2 ( n = 6–8 mice/group). Immunofluorescence staining (400×, merged) for PDGF‐Rα (red, white arrows; blue: DAPI) with decreased stain from the septal crests in lungs of ventilated PDGF‐Rα +/− (lower panel) and WT (upper panel) mice undergoing MV‐O 2 . Quantitative analysis of the immunofluorescence images showed reduced number of PDGF‐Rα + myofibroblasts located at the septal crests (presented myofibroblasts number per 100 septal crests; 10 fields of view in PDGF‐Rα and α‐smooth muscle actin co‐stained sections/animal, 4 animals/group). Immunoblot analysis of PDGF‐Rα (G) and its downstream proteins JAK‐2 (H) and STAT‐3 (I) showing a significant reduction in protein level in PDGF‐Rα +/− neonatal mice in contrast to WT mice after MV‐O 2 for 8 h ( n = 3 mice/group). PDGF‐Rα levels are displayed as fold change of control. Panels (H) and (I) are from same blot hence having same β‐actin bands. Data information: In (B–D) and (F–I), data are presented as mean ± SD. ** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: PDGF ‐Rα haploinsufficiency drives the air sac pathology of nCLD in neonatal mice undergoing MV ‐O 2 Representative lung tissue sections (200×) from 5–8‐day‐old PDGF‐Rα +/+ (WT) and PDGF‐Rα +/− mice after 8 h of MV‐O 2 showing increased air space size compared to respective controls (O 2 ‐control) spontaneously breathing 40% O 2 for 8 h. Quantitative analysis of lung tissue sections showed increased alveolar area after 8 h of MV‐O 2 in PDGF‐Rα +/− mice, whereas no significant change was observed in WT mice when compared to respective controls ( n = 6–11 mice/group). Radial alveolar counts (alveolar number) in lung tissue sections from WT and PDGF‐Rα +/− mice were reduced after 8 h of MV‐O 2 when compared to respective controls ( n = 6–11 mice/group). Septal density was significantly reduced in PDGF‐Rα +/− mice when compared to WT littermates after 8 h of MVO 2 ( n = 6–8 mice/group). Immunofluorescence staining (400×, merged) for PDGF‐Rα (red, white arrows; blue: DAPI) with decreased stain from the septal crests in lungs of ventilated PDGF‐Rα +/− (lower panel) and WT (upper panel) mice undergoing MV‐O 2 . Quantitative analysis of the immunofluorescence images showed reduced number of PDGF‐Rα + myofibroblasts located at the septal crests (presented myofibroblasts number per 100 septal crests; 10 fields of view in PDGF‐Rα and α‐smooth muscle actin co‐stained sections/animal, 4 animals/group). Immunoblot analysis of PDGF‐Rα (G) and its downstream proteins JAK‐2 (H) and STAT‐3 (I) showing a significant reduction in protein level in PDGF‐Rα +/− neonatal mice in contrast to WT mice after MV‐O 2 for 8 h ( n = 3 mice/group). PDGF‐Rα levels are displayed as fold change of control. Panels (H) and (I) are from same blot hence having same β‐actin bands. Data information: In (B–D) and (F–I), data are presented as mean ± SD. ** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Mouse Assay, Immunofluorescence, Staining

    Model for how attenuated PDGF signaling and positive pressure ventilation interact to produce the distinct phenotypic manifestations of nCLD MV‐O 2 in vivo , a combination of O 2 , that is, oxygen and stretch (purple arrow) and/or TGF‐β alone or in combination with mechanical stretch in vitro (yellow arrow), reduces platelet‐derived growth factor receptor α (PDGF‐Rα) levels and its downstream signaling through JAK‐2 and STAT‐3 in the pulmonary myofibroblast (MFB). This reduction in turn abrogates vascular endothelial growth factor expression (VEGF‐A and VEGF‐R2), leading to increased apoptosis in pulmonary endothelial cells (EC). Whereas myofibroblast migration is diminished through reduced RAS and pERK/ERK signaling, stretch alone increases their proliferation, hence depicting the differential effect of the most important denominators of nCLD development in the premature lung undergoing MV‐O 2 . Application of PDGF‐A to premature lung increases PDGF‐Rα levels in an AKT‐dependent manner in turn activating the downstream cascade through JAK‐2, STAT‐3 signaling. This then activates VEGF‐A secretion and VEGF‐R2 activity reducing apoptosis in endothelial cells (ECs).

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Model for how attenuated PDGF signaling and positive pressure ventilation interact to produce the distinct phenotypic manifestations of nCLD MV‐O 2 in vivo , a combination of O 2 , that is, oxygen and stretch (purple arrow) and/or TGF‐β alone or in combination with mechanical stretch in vitro (yellow arrow), reduces platelet‐derived growth factor receptor α (PDGF‐Rα) levels and its downstream signaling through JAK‐2 and STAT‐3 in the pulmonary myofibroblast (MFB). This reduction in turn abrogates vascular endothelial growth factor expression (VEGF‐A and VEGF‐R2), leading to increased apoptosis in pulmonary endothelial cells (EC). Whereas myofibroblast migration is diminished through reduced RAS and pERK/ERK signaling, stretch alone increases their proliferation, hence depicting the differential effect of the most important denominators of nCLD development in the premature lung undergoing MV‐O 2 . Application of PDGF‐A to premature lung increases PDGF‐Rα levels in an AKT‐dependent manner in turn activating the downstream cascade through JAK‐2, STAT‐3 signaling. This then activates VEGF‐A secretion and VEGF‐R2 activity reducing apoptosis in endothelial cells (ECs).

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: In Vivo, In Vitro, Derivative Assay, Expressing, Migration, Activity Assay

    Similar TGF ‐β activation with increased apoptosis in lungs of newborn PDGF ‐Rα haploinsufficient mice after MV ‐O 2 for 8 h Immunoblot analysis showing similar pSMAD levels in whole lung homogenate of newborn WT as well as PDGF‐Rα +/− mice ( n = 3 mice/group). Representative immunofluorescence image showing similar pSMAD levels (red) in both newborn WT (upper panel) and PDGF‐Rα +/− mice (lower panel). α‐SMA is in green, and nucleus is stained with DAPI (blue). Quantification of the image showing pSMAD‐2 area to 100 nuclei ( n = 3–4 mice/group, 4 sections/mice, 10 images/section). Increased apoptosis quantified by cleaved caspase‐3 + nuclei normalized to 100 nuclei in newborn PDGF‐Rα +/− compared to WT mice ( n = 4 mice/group). Data information: In (A–C), data are presented as mean ± SD. Statistical test is two‐tailed unpaired Student's t ‐test (* P = 0.0159).

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Similar TGF ‐β activation with increased apoptosis in lungs of newborn PDGF ‐Rα haploinsufficient mice after MV ‐O 2 for 8 h Immunoblot analysis showing similar pSMAD levels in whole lung homogenate of newborn WT as well as PDGF‐Rα +/− mice ( n = 3 mice/group). Representative immunofluorescence image showing similar pSMAD levels (red) in both newborn WT (upper panel) and PDGF‐Rα +/− mice (lower panel). α‐SMA is in green, and nucleus is stained with DAPI (blue). Quantification of the image showing pSMAD‐2 area to 100 nuclei ( n = 3–4 mice/group, 4 sections/mice, 10 images/section). Increased apoptosis quantified by cleaved caspase‐3 + nuclei normalized to 100 nuclei in newborn PDGF‐Rα +/− compared to WT mice ( n = 4 mice/group). Data information: In (A–C), data are presented as mean ± SD. Statistical test is two‐tailed unpaired Student's t ‐test (* P = 0.0159).

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Activation Assay, Mouse Assay, Immunofluorescence, Staining, Two Tailed Test

    Pronounced effect of TGF ‐β on pulmonary myofibroblasts from PDGF ‐Rα +/− mice and in concert with mechanical stretch on both mice and human myofibroblasts TGF‐β application (Th1) to myofibroblasts (MFBs) isolated from neonatal PDGF‐Rα +/− mice reduced PDGF‐Rα (A), JAK‐2 (B), STAT‐3 (C), VEGF‐A (D), and pERK/ERK (E) protein levels when compared to control ( n = 3–6 mice/group). Panels (A, E) and (B, C) are from same blot hence having same β‐actin bands. Reduced migration assessed by Boyden chamber in myofibroblasts (MFBs) isolated from PDGF‐Rα +/− mice compared to WT mice ( n = 5 mice/group). TGF‐β application in combination with mechanical stretch (S+Th1) in myofibroblasts (MFBs) isolated from neonatal WT mice showed reduced PDGF‐Rα (G) and VEGF‐A (H) protein levels as well as migratory RAS (I) and pERK/ERK (J) protein levels when compared to control myofibroblasts as assessed by immunoblot assay ( n = 6–9 mice/group). TGF‐β application (Th1) as an additional dose (Th2) on stretched myofibroblasts (MFBs) from WT mice reduced migration as assessed by Boyden chamber assay ( n = 5 mice/group). TGF‐β application in combination with stretch (S+Th1) reduced PDGF‐Rα protein levels in fibroblasts (hMFBs) isolated from tracheal aspirates of nCLD patients when compared to control (C) or stretched (S) myofibroblasts (L) and as an additional dose (Th2) to stretched fibroblasts reduced migration (M) ( n = 3–6 patients/group). Data information: Values are normalized to the respective controls except for (B–D). Data are presented as mean ± SD. Statistical test used in (A, D–F) is two‐tailed and in (B, C) is one‐tailed Student's t ‐test or Mann–Whitney test ( P = 0.004–0.05) and in (G–M) is ordinary one‐way ANOVA with Bonferroni's correction ( P = 0.0001–0.04). *** P

    Journal: EMBO Molecular Medicine

    Article Title: Attenuated PDGF signaling drives alveolar and microvascular defects in neonatal chronic lung disease

    doi: 10.15252/emmm.201607308

    Figure Lengend Snippet: Pronounced effect of TGF ‐β on pulmonary myofibroblasts from PDGF ‐Rα +/− mice and in concert with mechanical stretch on both mice and human myofibroblasts TGF‐β application (Th1) to myofibroblasts (MFBs) isolated from neonatal PDGF‐Rα +/− mice reduced PDGF‐Rα (A), JAK‐2 (B), STAT‐3 (C), VEGF‐A (D), and pERK/ERK (E) protein levels when compared to control ( n = 3–6 mice/group). Panels (A, E) and (B, C) are from same blot hence having same β‐actin bands. Reduced migration assessed by Boyden chamber in myofibroblasts (MFBs) isolated from PDGF‐Rα +/− mice compared to WT mice ( n = 5 mice/group). TGF‐β application in combination with mechanical stretch (S+Th1) in myofibroblasts (MFBs) isolated from neonatal WT mice showed reduced PDGF‐Rα (G) and VEGF‐A (H) protein levels as well as migratory RAS (I) and pERK/ERK (J) protein levels when compared to control myofibroblasts as assessed by immunoblot assay ( n = 6–9 mice/group). TGF‐β application (Th1) as an additional dose (Th2) on stretched myofibroblasts (MFBs) from WT mice reduced migration as assessed by Boyden chamber assay ( n = 5 mice/group). TGF‐β application in combination with stretch (S+Th1) reduced PDGF‐Rα protein levels in fibroblasts (hMFBs) isolated from tracheal aspirates of nCLD patients when compared to control (C) or stretched (S) myofibroblasts (L) and as an additional dose (Th2) to stretched fibroblasts reduced migration (M) ( n = 3–6 patients/group). Data information: Values are normalized to the respective controls except for (B–D). Data are presented as mean ± SD. Statistical test used in (A, D–F) is two‐tailed and in (B, C) is one‐tailed Student's t ‐test or Mann–Whitney test ( P = 0.004–0.05) and in (G–M) is ordinary one‐way ANOVA with Bonferroni's correction ( P = 0.0001–0.04). *** P

    Article Snippet: Primary neonatal mouse myofibroblasts were transfected with either 100 nM specific siRNA against PDGF‐Rα (Santa Cruz Biotechnology, Inc., Germany #sc‐29444) or 100 nM control siRNA B (Santa Cruz Biotechnology #sc‐44230) suspended in TurboFect (Thermo Fisher Scientific, Waltham, MA, USA #R0531) or remained untreated controls in siRNA transfection media (Santa Cruz Biotechnology, Inc., #sc‐36868).

    Techniques: Mouse Assay, Isolation, Migration, Boyden Chamber Assay, Two Tailed Test, One-tailed Test, MANN-WHITNEY

    Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Synthesis of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Conditions in each step: (i) H 2 SO 4 /HNO 3 ; (ii) SOCl 2 ; (iii) NH 2 (CH 2 ) 2 NH-Boc/THF and HCl/EA; and (iv) EDC/Boc-Arg(Tos)-Gly-Asp(OMe)-Val-OH, NaOH/MeOH, and TFA/TfOH. Abbreviations: siRNA, small interfering RNA; THF, tetrahydrofuran; EA, ethyl alcohol; EDC, N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine; TFA, trifluoroacetic acid; TfOH, trifluoromethanesulfonic acid; ND, nanodiamond.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Antiproliferation effect of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA against MCF-7 cells at different concentrations. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Viability of MCF-7 cells after treatment with different concentrations of NDCONH(CH 2 ) 2 NH-VDGR, survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/NC, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, Lipo/NC, and Lipo/survivin-siRNA for 48 h. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The DSC curves of NDCONH(CH 2 ) 2 NH-VDGR and different concentrations of NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Abbreviations: DSC, differential scanning calorimetry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: AFM images of mouse plasma alone ( A ), ND in mouse plasma ( B ), NDCONH(CH 2 ) 2 NH-VDGR in mouse plasma ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA in mouse plasma ( D ). Abbreviations: AFM, atomic force microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Microscopy, Small Interfering RNA

    The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The apoptosis of MCF-7 cells induced by survivin-siRNA ( B ), NDCONH(CH 2 ) 2 -NH-VDGR ( C ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( D ). Cells treated with PBS solution served as control ( A ) (n=3). Abbreviations: siRNA, small interfering RNA; PBS, phosphate-buffered saline; FITC-A, fluorescein isothiocyanate–Annexin V; PI-A, propidium iodide–Annexin V.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Confocal images of control ( A ), cells treated with naked FAM-survivin-siRNA ( B ), and cells treated with NDCONH(CH 2 ) 2 NH-VDGR/FAM-survivin-siRNA ( C ). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cumulative releasing percentage of survivin-siRNA from NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, ND/survivin-siRNA, and naked survivin-siRNA in TE buffer (n=3). Abbreviations: siRNA, small interfering RNA; ND, nanodiamond; TE, Tris–ethylene diamine tetraacetic acid.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tyndall effect of ND ( B ) and NDCONH(CH 2 ) 2 NH-VDGR ( C ). Water ( A ) served as control. The zeta potential of ND ( D ), NDCONH(CH 2 ) 2 NH-VDGR ( E ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( F ). Abbreviations: ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: The cell percentage of blank, naked survivin-siRNA, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA, and Lipo/survivin-siRNA group in each period of cell cycle (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: SEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: SEM, scanning electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Electron Microscopy, Small Interfering RNA

    Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Agarose gel retardation of naked survivin-siRNA and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA at different N/P ratios. Abbreviations: siRNA, small interfering RNA; N, negative; P, positive.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin-mRNA expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation

    Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor weights of the normal control (NS), survivin-siRNA group, NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group, and DOX group (n=10). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin; SD, standard deviation.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA, Standard Deviation

    Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Tumor tissues from normal control (NS) group ( A ), survivin-siRNA group ( B ), NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group ( C ) and DOX group ( D ). Abbreviations: siRNA, small interfering RNA; DOX, doxorubicin.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: MS spectra of organ homogenate in NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA group. RGDV ( m / z ): 444.22012 [M-H] − . Abbreviations: MS, mass spectrometry; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Mass Spectrometry, Small Interfering RNA

    TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: TEM images of ND ( A ), NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: TEM, transmission electron microscopy; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy, Small Interfering RNA

    Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Survivin protein expression of MCF-7 cells treated with NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA. Data are presented as the average ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation; ELISA, enzyme-linked immunosorbent assay; NC, normal control.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Expressing, Small Interfering RNA, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Journal: International Journal of Nanomedicine

    Article Title: Gene-silencing effects of anti-survivin siRNA delivered by RGDV-functionalized nanodiamond carrier in the breast carcinoma cell line MCF-7

    doi: 10.2147/IJN.S117611

    Figure Lengend Snippet: Particle size and PDI of ND ( A ) and NDCONH(CH 2 ) 2 NH-VDGR ( B ), and NDCONH(CH 2 ) 2 NH-VDGR/survivin-siRNA ( C ). Abbreviations: PDI, polydispersity index; ND, nanodiamond; siRNA, small interfering RNA.

    Article Snippet: Boc-HN-(CH2 )2 -NH2 was purchased from GL Biochem Ltd. Anti-survivin siRNA and fluorescein-labeled survivin-siRNA (FAM-survivin-siRNA) were purchased from GenePharma Co., Ltd (Shanghai, People’s Republic of China).

    Techniques: Small Interfering RNA

    Nanog overexpression or suppression of MAPK/ERK signaling rescues Tet1 KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control siRNA and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

    doi: 10.1093/nar/gkr1253

    Figure Lengend Snippet: Nanog overexpression or suppression of MAPK/ERK signaling rescues Tet1 KD phenotype. ( A ) Western blot analysis showing Nanog overexpression with HA-tag. ( B ) AP staining of E14Tg2a mESCs, with and without Nanog overexpression, transfected with control siRNA and Tet1 siRNA #1. Cells were cultured in normal ESC medium, and AP staining was performed 96 h after transfection. ( C ) Relative mRNA levels of selected mESC pluripotency genes and differentiation marker genes in control and Tet1 KD mESCs in 2i medium. Oct4GiP cells were transfected with control siRNA or Tet1 siRNA #1 at 50 nM in 24-well plates in 2i-medium (which inhibits MAPK/ERK and Gsk-3b signaling) and cells were harvested 96 h after transfection. The mRNA levels in control cells are set as one. Data are normalized to Actin . Error bars represent SEM of three experiments.

    Article Snippet: RNAi experiments were performed using indicated individual siRNAs: Tet1 siRNA #1 (Invitrogen, MSS284895), Tet1 siRNA #2 (Invitrogen, MSS284897), Tet1 siRNA #3 (Dharmacon, D-062861-01), Tet1 siRNA #4 (Dharmacon, D-062861-02), Tet1 siRNA #5 (Dharmacon, D-062861-03), Tet1 siRNA #6 (Dharmacon, D-062861-04) and control siRNA duplex targeting firefly luciferase (Dharmacon, 5′-CGTACGCGGAATACTTCGA).

    Techniques: Over Expression, Western Blot, Hemagglutination Assay, Staining, Transfection, Cell Culture, Marker