Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) E2012 and E2012-BPyne structures. (b) Cell membranes were photolabeled with E2012-BPyne and then clicked with TAMRA-azide and analyzed (left: Coomassie blue; right: in-gel fluorescence). (c) Biotinylated proteins were captured by E2012-BPyne and analyzed by WB. (d) IFITM3 co-immunoprecipitated with γ-secretase subunits using anti-PS1. (e) IFITM3 was co-purified with γ-secretase subunits by GY6 or 163-BP-L-biotin. (f) An interaction (red) between PS1 and IFITM3 was determined by in situ PLA in mouse primary neurons (bottom panel). Negative controls using PS1 or IFITM3 alone (top two panels), dapi (blue), F-actin (green), scale bar = 50μm. (g) Labeling of IFITM3 by E2012-Bpyne in WT and dKO MEF cells. All WB and IF images are representative of three independent experiments (except b: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Fluorescence, Immunoprecipitation, Purification, In Situ, Labeling

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) LC-MS/MS analysis of the 15 kDa band identified four peptides that match with human IFITM3. (b) WB analysis of E2012-BPyne (500 nM) labeled PS1-NTF protein. (c) Structures of imidazole GSMs, acid GSM and GSIs. (d) WB analysis of E2012-BPyne labeled proteins in the absence or presence of imidazole GSMs, acid GSM and GSIs. Labeled proteins were captured and analyzed by WB for IFITM3. (e) Structures of GY6 and 163-BP-L-biotin. (f) IFITM3 co-immunoprecipitates with γ-secretase subunits. CHAPSO solubilized cell membranes were immunoprecipitated with anti-IFITM3 antibody and probed with antibodies against PS2-CTF and Pen-2. Rabbit IgG was used as a negative control. (g) IFITM3 does not co-immunoprecipitate with SPP. CHAPSO solubilized cell membranes were immunoprecipitated with a monoclonal anti-IFITM3 antibody (9D11) and probed with antibodies against SPP and IFITM3. Mouse IgG was used as a negative control. (h) Analysis of the total protein level in WT MEF or PS1/2 double KO MEF cells. The same amount of membrane proteins was loaded and analyzed by Western blotting. ( i ) IFITM3 mRNA expression levels were measured by RT-PCR in WT MEF or PS1/2 double KO MEF cells (n=6). All WB images and graphs are representative of three independent experiments (except; a/d: 2 replicates). Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Immunoprecipitation, Negative Control, Membrane, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) IFITM3 KD by siRNA (n=3) in HEK-APP WT cells was confirmed by WB. Scramble siRNA (SC, n=3), a negative control. (b) IFITM3 KD reduces secreted Aβ40 (n=6, ****p<0.0001) and 42 (n=6, ****p<0.0001). Aβ levels were calculated as % of SC. (c) IFITM3 was KO of U138 cells, and empty vector (EV) was used as control. IFITM3 was reintroduced by transient transfection in both EV and KO cell lines. IFITM3 expression was confirmed by WB. (d) Effect of KO and rescue of IFITM3 on γ-secretase activity for Aβ40 (n=3, *p= 0.0249, ****p<0.0001) and 42 (n=3, *p=0.0359, **p=0.0025) cleavage. (e) Comparison IC 50 of GSM E2012-BPyne against γ-secretase for Aβ40 (n=6, **p= 0.0017) and Aβ42 (n=6, ns) cleavages in U138 EV and KO cells. All WB images and graphs are representative of three independent experiments (except; e: contains data from 2 experimental replicates of n=3). Graphs are mean ± SD. Ns, not significant, two-sided Student’s t-test (except; d: one-way ANOVA and Fishers LSD test).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Negative Control, Plasmid Preparation, Control, Transfection, Expressing, Activity Assay, Comparison

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Quantification of WB showed that IFITM3 KD did not change protein expression levels of APP, Nct and PS1-NTF in HEK-APP WT cells (n=3). (b) Schematic representation of cell-free γ-secretase assay. γ-Secretase is incubated with a recombinant APP substrate in the presence of 0.25% CHAPSO. Cleaved Aβ40 and 42 species are measured with cleavage specific antibodies and AlphaLISA technology. ( c ) Schematic model showing different GSM and GSI binding sites in γ-secretase: E2012 (imidazole GSM), GSM-1 (acid GSM), and L458 (transition state analogue inhibitor, GSI). (d-f) Comparison of IC 50 of (d) GSM-25 (EV: n=9, KO: n= 8) for Aβ40 (****p<0.0001) and Aβ42 (ns), (e) GSM-1 (n=6) for Aβ40 (**p=0.0039) and Aβ42 (ns), and (f) L458 (n=3) for Aβ40 (ns) and Aβ42 (ns) cleavages in the U138 EV or KO cell lines (n≥3). (g) IFITM3 knockdown (KD) does not affect expression of γ-secretase subunits. IFITM3 was knocked-down by siRNA (6 pmol, n=3) in HEK-NotchΔE cells and scramble siRNA (SC, n=3) was used as a negative control. Cell lysates were probed by antibodies against Nct, PS1-NTF and IFITM3. β-Actin was used as a loading control. (h) Effect of IFITM3 KD on γ-secretase activity. IFITM3 KD increased γ-secretase cleaved product NICD, analyzed by WB. Cell lysates were probed by antibodies against c-myc (NotchΔE) and NICD and a representative quantification of NICD (n=8, ***p=0.001) is shown (lower panel). (i) Cell based NICD AlphaLISA assay (left panel) revealed an increase in NICD production with IFITM3 KD. Quantification of NICD (n=8, ***p=0.001) is shown in the right panel. (j) Effect of IFITM3 KO on γ-secretase activity. KO cells lines have increased γ-secretase activity as compared to the EV cell line. The NICD cleavage in vitro was measured by AlphaLISA assay (n=3,**p=0.0096). All WB images and graphs are representative of three independent experiments Graphs are mean ± SD. ns, not significant, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Incubation, Recombinant, Binding Assay, Comparison, Knockdown, Negative Control, Control, Activity Assay, In Vitro

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Protein levels of γ-secretase subunits and IFITM3 in pooled membranes from 4- and 28-month-old WT mouse brains (n=5 mice per age and sex group except n=4 for 28 female, female: **p=0.0014, male: **p=0.0065). Quantified as percent of 4-month female level. (b) γ-Secretase activity for Aβ40 (female: p=***0.0002, male: **p=0.0095) and Aβ42 (female: p=**0.0024, male: **p=0.0088) production in vitro from pooled membrane. (c) Solubilized membranes were captured by 163-BP-L-biotin and then analyzed for γ-secretase and IFITM3 levels. (d) WB for PS1-NTF and IFITM3 (left panel, representative mice shown) in membranes of 18-month-old WT (n=4) and IFITM3−/− (n=5) mouse brains. γ-Secretase activity for Aβ40 (***p=0.0004) and 42 (****p=<0.0001) cleavage (right panels). (e) APP, Nct, PS1-NTF and IFITM3 in membranes from 3- and 12-month-old WT and 5xFAD mice. Quantified as percent of 3-months-old WT (n=5 per group except n=4 for WT at 12 months)(3moWT-12mo5X: ****p<0.0001, 12moWT-12mo5X: ***p=0.004, 3mo5X-12mo5X: ***p=0.0003). (f) WB for PS1-NTF and IFITM3 (left panel) in membranes from 4-month-old 5xFAD (n=3) and IFITM3−/−; 5xFAD (n=3) mouse brains. γ-Secretase activity in vitro for Aβ40 (***p=0.0003) and 42 (**p=<0.0011) cleavage (right panels). (g) Fluorescence microscopy of amyloid plaques in cortex and hippocampus (Thioflavin-S, green) in 4-month-old PFA perfused mice (5xFAD: n=4, IFITM3−/−; 5xFAD: n=5). Scale bar = 300μm (left), 200μm (right). Number of plaques per mm 2 of tissue was calculated throughout the brain and averaged (cor (cortex): *p=0.0109, hip (hippocampus): **p=0.0026). All WB and IF images and graphs are representative of three independent experiments (except; c and quantifications in e: 2 replicates and g: every 1/6 th section throughout the brain). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). Ns, not significant, two-sided Student’s t-test (except; e: two-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Activity Assay, In Vitro, Membrane, Fluorescence, Microscopy

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) WBs for Nct and PS1-NTF were quantified by Odyssey imaging (n=5 mice pooled per group, except n=4 for 28F, all=ns). (b) Effect of aging on subcellular localizations of IFITM3. A hemibrain from male wild-type C57BL/6 mouse at 4 and 28 months (n=1 per group) were homogenized and layered on iodixanol gradient (2.5 – 30 %). Fractions were collected from the top and resolved by WBs for γ-secretase, IFITM3 and different subcellular markers. (c) WBs for APP, Nct, and PS1-NTF were quantified by Odyssey imaging (n=5 mice per group except n=4 for WT at 12 months). APP: 3moWT-3mo5X: ****p<0.001, 3mo5X-12mo5X: ****p<0.0001, 12moWT-12mo5X: ****p<0.0001). Nct: 3moWT-12moWT: ****p<0.001, 3mo5X-12mo5X: ****p<0.001, 12moWT-12mo5X: ****p<0.001. PS1-NTF: 3moWT-12mo5X: ***p=0.0004, 12moWT-12mo5X: ****p<0.001. ( d ) Immunostaining of IFITM3 in mouse brains. Fluorescence microscopy of IFITM3 expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of cortex, hippocampus and subiculum (left to right) show IFITM3 (green) and DAPI (blue). Scale bar = 1000μm, 200μm, 100μm (left to right). Total IFITM3 fluorescence area within the hippocampus and cortex of WT and 5XFAD was quantified using FIJI. Total IFITM3 was divided by tissue area and 5XFAD expression was normalized to average of WT (WT: n=7, 5XFAD: n=9)(cor (cortex): **p=0.0035, hip (hippocampus): ****p<0.0001 ). (e) IFITM3 expression in astrocytes and microglia is upregulated in 5XFAD mice compared to WT mice. Fluorescence microscopy of IFITM3, GFAP (top) and Iba1 (bottom) expression in 12-month-old PFA perfused mice (WT, upper panel, 5XFAD, lower panel). Representative images of the hippocampus and cortex show IFITM3 (red), GFAP (green – top), Iba1 (green - bottom), and DAPI (blue), scale bar = 500μm. Inset panels (left to right) show GFAP or Iba1 (green), IFITM3 (red) and merge. Scale bar = 50μm. All WB images and graphs are representative of three independent experiments (except; b: 2 replicates). Graphs are mean ± SD. ns, not significant, , two-sided Student’s t-test, (except; c: one-way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Imaging, Immunostaining, Fluorescence, Microscopy, Expressing

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) The expression profiles of IFITM3 in human control (n=76) and LOAD (n=80) from temporal cortex (**p=0.002)(Mayo Clinic cohort). (b) IFITM3 mRNA expression levels in control and LOAD samples (LOAD: n=18 and control: n=10, **p=0.0056). (c) The protein levels of γ-secretase subunits and IFITM3 in human brain membranes (LOAD: n=18 and control: n=10, *p=0.0127). IFITM3 quantified as relative intensity. (d) IFITM3 expression (left) between control (n=10), LOAD-L (n=10) and LOAD-H (n=8)(****p<0.0001). γ-Secretase activity for Aβ40 (**p=0.0037, ***p=0.0007) and 42 (**p=0.0036, ***p=0.0002) cleavage between groups. (e) Primary mouse neurons were treated with control (n=2), 10ng/mL (n=3, ***p=0.0005) or 100 ng/mL (n=3, ***p=0.0003) of IFN-γ, membranes were probed for γ-secretase and IFITM3 (left panel, quantified: right panel). (f) Quantification of secreted Aβ40 from 10ng/mL (n=8, **p=0.0010) and 100 ng/mL (n=8, **p=0.0026) IFN-γ treated neurons Aβ42 from 10ng/mL (n=8, ***p=0.0006) and 100 ng/mL (n=8, ***p=0.0003) IFN-γ (n=8). (g) γ-Secretase activity for Aβ40 (n=6, ***p=0.0007) and 42 (n=6, ***p=0.006) cleavage from IFN-γ treated neuron membranes. (h) Photolabeled PS-NTF in neuronal membranes and quantified as % of control (control: n=10, 10ng/ml: n=5, **p=0.0023, 100ng/ml: n=7,****p<0.0001). (i) WB for IFITM3 and PS1-NTF primary human astrocytes treated with PBS, IL-6, or IL-1β (control: n=6, IL-6: n=4, **p=0.0014, IL-1β: n=3, *p=0.0103), data normalized to PBS. (j) γ-Secretase activity for Aβ40 (control: n=8 IL-6: n=7, **p=0.0098, IL-1β: n=9,****p<0.0001) and 42 (control: n= IL-6: n=7, *p=0.0467, IL-1β: n=9, ****p<0.0001) in astrocyte membrane. All WB images and graphs are representative of three independent experiments (except; b: 1 replicate, c/g: 2 replicates, d: γ-secretase activity graph contains data from two independent replicates, h: 5 replicates). Graphs are mean ± SD. Ns, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test.

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Activity Assay, Membrane

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Spearman’s correlation of mRNA expression of human IFITM3 gene with age was analyzed in the cortex (n=158) and hippocampus (n=123) of normal human brains using the Genotype-Tissue Expression (GTEx) cohort.. (b) mRNA expression in non-demented subject control (n=10) and LOAD samples (n=18) of MAP2 (ns), GFAP (**p=0.0046), and AIF1 (ns) were measured, which were used in – . (c) Expression profiles of MAP2 (ns), GFAP (****p<0.0001), and AIF1 (ns) in the temporal cortex of human control (n=76) and LOAD samples (n=80) using the Mayo Clinic cohort data. Correlation analyses were carried out and p values were calculated. The protein levels of Nct (****p<0.0001) and PS1-NTF (**p=0.0042) in human brain membranes (control and LOAD). The samples were analyzed by WB and quantified (n=10 and 18, respectively). Signal was normalized to HeLa cell membrane. (e-f) IFITM3 SNP Genotypes. (e) Allelic discrimination plot depicting rs34481144 genotype calls for control (n=9), LOAD-L (n=10), and LOAD-H (n=8) brain samples. The axes show delta Rn values obtained from TaqMan SNP genotyping analysis. Samples without genomic DNA were used as non-template controls (shown as black squares in the left lower quadrant, n=2). (f) Allele frequency of rs34481144 genotype in control (n=9) and LOAD (n=18). ( g ) mRNA level of IFITM3 gene in four types of EGFP/L10a-expressing mouse hippocampal neurons (GAD2 (glutamate decarboxylase 2), CCK (cholecystokinin), PV (parvalbumin), and CORT (cortistatin) expressing GABAergic neurons)(n=4 per group, GAD2-PV: ***p=0.0003, CCK-PV: ****p<0.0001, CCK-Cort *p=0.0363, PV-Cort: **p=0.0059). ( h ) mRNA levels of IFITM3 in human iPSC-derived neurons (n=4) and human primary astrocytes (n=3) were measured by qPCR (****p<0.0001). ( i-j ) Human iPSC-derived neurons (i) and human primary astrocytes (j) were stained for IFITM3 with MAP2 (neuronal marker) or S100β (astrocyte marker). DAPI was used for nucleus staining. Scale bar = 200 and 500 μm. ( k ) Induction of IFITM3 by IFN-α in primary neurons. E16 mouse primary neurons were treated with 100 ng/ml of IFN-α at DIV12 for 24 hours. The protein levels of γ-secretase and IFITM3 were analyzed by WB (n=4 per group). β-Tubulin III was used as a loading control. ( l ) Effect of IFITM3 induction on γ-secretase activity for Aβ40 (*p=0.0116) and Aβ42 (*p=0.0319) activity. Membranes from primary neurons were incubated with the recombinant APP substrate C100-ΔID-FLAG and γ-secretase activity (Aβ cleavage rate) was assayed by human Aβ three-plex MSD kits (n=12, 10). ( m ) JC8 whole cell photolabeling. Neuronal membranes were photolabeled with JC8 in the absence or presence of L458 and analyzed by anti-PS1-NTF antibody. Photolabeled PS1-NTF protein level was quantified by Odyssey imaging (n=3, *p=0.0210). (n) Spearman’s correlation between the expression level of IFITM3 and viruses. In the Brodmann Area 22 (BA-22 region) in the Mount Sinai Brain Bank (MSBB) cohort, the expression level of IFITM3 is positively correlated with the expression level of the human herpesvirus-6B (HHV-6B) (rho=0.248, p=0.044, n=66). In the Brodmann Area 36 (BA-36 region), the expression level of IFITM3 is positively correlated with the expression of hepatitis C virus genotype 4 (rho=0.255, p=0.033, n=70). All WB images and graphs are representative of two independent experiments (except; b/e: 1 replicate, h: data pooled from 2 experiments, k: 3 replicates, l: data pooled from 4 experiments). Bar graphs are mean ± SD. Violin plots represent median (middle line) and interquartile range (outer lines). ns, not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, two-sided Student’s t-test (except; g: One-Way ANOVA followed by Tukey).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Expressing, Control, Membrane, Derivative Assay, Staining, Marker, Activity Assay, Incubation, Recombinant, Imaging, Virus

Journal: Nature

Article Title: Innate immunity protein IFITM3 modulates γ-secretase in Alzheimer disease

doi: 10.1038/s41586-020-2681-2

Figure Lengend Snippet: (a) Schematic model showing L458 binding to the subsites (S2-S3’) in the active site of γ-secretase. (b) Photolabeling of IFITM3 and PS1 by four inhibitors. (c) Photolabeling of IFITM3 and PS1 by L505 in human brains (control: n=4, LOAD-L: n=5, LOAD-H: n=5). (d) Pearson’s correlation between γ-secretase activity and L505 labeled IFITM3 (Fig. 5c) in LOAD samples (n=10). (e) Double cross-linking of γ-secretase and IFITM3 by the dual probe, L631. WB reveals multiple protein complex species containing IFITM3, PS1-NTF, PS1-CTF, IFITM3 homodimer, IFITM3-PS1 and PS1-NTF-CTF heterodimers. (f) Schematic representation of the interaction between IFITM3 and γ-secretase, IFITM3 is near the active site and can be crosslinked with PS1-NTF. (g) IFITM3 connects infections and innate immunity with Aβ production and AD risk. (A) Pathogenic challenge, or other inflammatory conditions, induce the release of proinflammatory cytokines from astrocytes and microglia. (B) Cytokines upregulate IFITM3 expression in neurons and astrocytes that potentiates γ-secretase, increasing Aβ production. (C) As part of an innate immune response, Aβ acts as an antimicrobial or antiviral peptide. In turn, Aβ accumulation also triggers AD pathology. All WB images and graphs are representative of three independent experiments (except; c: 2 replicates).

Article Snippet: Plasmid DNAs for human IFITM3 (OriGene) were cloned into the pcDNA3.1 vector.

Techniques: Binding Assay, Control, Activity Assay, Labeling, Expressing

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 1. Generation of LMO2 knockdown (KD) or overex- pressing cells. Human umbilical vein endothelial cells (HUVECs) passage 4 (P4) were infected with lentiviral scramble short hairpin RNA (shRNA) virus or LMO2 shRNA virus and selected with puromycin (1 lg/mL) for 14 consecutive days to generate stable KD cells. HUVECs P4 were infected with lentiviral particles encoding LMO2 open reading frame (ORF) or virus without the ORF and selected with puromycin to generate stable overexpres- sion (OE) or control (CT) cell lines. A, reverse transcription polymerase chain reaction (RT-PCR) examination of LMO2 mRNA level of KD cells. B, Western blot examination of LMO2 protein level in KD cells. C, RT-PCR examination of LMO2 mRNA level in OE cells. D, Western blot examination of LMO2 protein level in OE cells. Data are presented as meanSEM (n=3). *P<0.05 vs CT.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Knockdown, Infection, shRNA, Virus, Control, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 2. LMO2 knockdown (KD) reduced cell proliferation and hindered G1/S transition. A, Cell growth curve. B, Cell cycle analysis by propidium iodide (PI) staining. C, Quantification of cell fractions in G1, S, and G2/M phases. D, Western blot examination of G1/S transition-related genes. E and F, chromatin immunoprecipitation polymerase chain reaction analysis of LMO2 complex binding to CDK2 gene (+2700) and CDK4 gene (+232) in both control (CT) and LMO2 KD cells. Data are presented as meanSEM (n=3). *P<0.05 vs CT.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Knockdown, Cell Cycle Assay, Staining, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Control

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 3. LMO2 overexpression promoted endothelial G1/S transition. A, Cell cycle analysis by propidium iodide (PI) staining. B, Quantification of cell fractions in G1, S, and G2/M phases. C, mRNA level of G1/S-related genes. D, Western blot examination of G1/S- related genes. Data are presented as meanSEM (n=3). *P<0.05 vs control (CT).

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Over Expression, Cell Cycle Assay, Staining, Western Blot, Control

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 4. LMO2 knockdown (KD) impaired endothelial network formation, which can be rescued by exogenous transforming growth factor-b1 (TGF-b1). A, Network formation of control (CT) and LMO2 KD human umbilical vein endothelial cells (HUVECs). B, List of downregulated genes identified in LMO2 KD in comparison to CT HUVECs using angiogenesis polymerase chain reaction (PCR) array. C, Reverse transcription PCR (RT- PCR) of TGF-b1. D, ELISA for TGF-b levels in culture medium of CT and LMO2 KD cells. E, RT-PCR of TGF-b1 in CT and LMO2 overexpression HUVECs. F, Network formation of CT and LMO2 KD HUVECs with or without supplementation of TGF-b 10 ng/mL. G, Chromatin immunoprecipitation PCR analysis of LMO2 complex binding to TGF-b1 promoter (–901 and –4030 region) in both CT and LMO2 KD cells. Data are presented as meanSEM (n=3). *P<0.05 vs CT.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Knockdown, Control, Comparison, Polymerase Chain Reaction, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Over Expression, Chromatin Immunoprecipitation, Binding Assay

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 5. lmo2 gene expression increased following caudal fin resection and the regeneration process. A, To analyze changes in gene expression in the regenerating caudal fin, the amputated tissues from 15 fish were collected at day 0 (left panel) and regenerating distal tissues were resected and collected at 1, 2, or 5 days after the original resection (n=5 for each time point) (right panel, a fin at 2 days postresection). B, Gene expression of lmo2 in the regenerating fin. C, Gene expression of tie2 in the regenerating fin. Data are presented as meanSEM (n=5). *P<0.05 vs control.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Gene Expression, Control

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 6. lmo2 knockdown (KD) by vivo-morpholino (Mo) in zebrafish reduced prolifer- ating GFP+ cells. A, Adult Tg(fli1:egfp)y1 zebrafish fish (2.5 months old) received a retro- orbital injection of PBS solution containing lmo2 or control (CT) vivo-Mo, then the caudal fin was resected and injected with 5-bromo-20-deoxyuridine (BrdU), as shown in the diagram. B, At day 15, whole fish were enzymatically digested and cell suspension was analyzed by flow cytometry. Negative control was Wik (non-GFP) fish line not injected with BrdU. BrdU+ and GFP+ events are shown in CT and lmo2 vivo-Mo–injected fish. C and D, Diagrams showing the percentage of BrdU+GFP/GFP cells and BrdU+GFP+/GFP+ cells. E, Western blot of lmo2 expression at day 10 in zebrafish without vivo-Mo injection or injected with CT or lmo2 vivo-Mo injection. Data are presented as meanSEM (n=10). *P<0.05 vs CT.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Knockdown, Injection, Control, Suspension, Cytometry, Negative Control, Western Blot, Expressing

Journal: Journal of the American Heart Association

Article Title: LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration

doi: 10.1161/jaha.116.004117

Figure Lengend Snippet: Figure 7. lmo2 KD by vivo-morpholino (Mo) in zebrafish reduced caudal fin regeneration and impaired vascular regeneration. Adult Tg(fli1: egfp)y1 zebrafish fish (2.5 months old) were injected with lmo2 or control (CT) vivo-Mo, then the caudal fin was resected and fin regeneration was observed. A, Representative caudal fin images before resection and then at 0, 1, 2, and 5 days postresection (dpr) in fish injected with CT or lmo2 vivo-Mo. B, Quantification of the regeneration. The whole area of the caudal fin at day 0 before resection and the new fin tissue area in each fish from the new distal fin edge to the amputation plane were quantified at 0, 1, 2, and 5 dpr. Caudal fin regeneration for each fin at each time point was defined as percentage of regeneration=1009 (regenerated tissue area/original fin area amputated). C, Representative image showing decreased vasculature length in the regenerated fin in lmo2 KD fish compared with CT. The vertical white bar represents the length of the regenerated vasculature. D, Quantification of the regenerated vascular area in CT at 5 dpr. Data are presented as meanSEM (n=10). *P<0.05 vs CT.

Article Snippet: Lentiviral particles of control (CT) short hairpin RNA (shRNA) and LMO2 shRNA were from Santa Cruz Biotechnology Inc. CT noncoding and LMO2 open reading frame (ORF) OE lentiviral particles were from Applied Biological Materials Inc (Richmond, BC, Canada).

Techniques: Injection, Control

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 are required for VEGF-induced Akt-mTORC1 and Erk activation in mouse embryonic fibroblasts. (A-B) WT MEFs, Gαi1 or Gαi3 single knockout (SKO) MEFs, Gαi2 SKO MEFs, and Gαi1/3 double knockout (DKO) MEFs were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of listed proteins. (C-D) DKO MEFs were transiently transfected with the vector encoding Ad-Gαi1, Ad-Gαi3 or empty vector (pDC315); VEGF (“-A”, 25 ng/mL)-induced signaling was tested. The same sets of lysate samples were run in sister gels to test different proteins (same for all figures). Indicated protein was quantified via ImageJ software (same for all figures). “n=5” means quantification from five replicate blot data (same for all figures). Bars stand for mean ± SD (same for all Figures).* P < 0.05 vs. WT MEFs. # P < 0.05 in (A). # P < 0.05 vs. Vector MEFs in (D).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Activation Assay, Knock-Out, Double Knockout, Western Blot, Transfection, Plasmid Preparation, Software

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 are required for VEGF-induced VEGFR2 endocytosis in mouse embryonic fibroblasts. (A-B) WT MEFs were treated with VEGF (“-A”, 25 ng/mL) for indicated times; associations between VEGFR2, p-VEGFR2, Ephrin-B2, Dab2, PAR-3, Gαi1 and Gαi3 were tested by co-immunoprecipitation (“IP”) assay. (C) WT and DKO MEFs were treated with VEGF (“-A”, 25 ng/mL) for 5 min;VEGFR2, p-VEGFR2, Ephrin-B2, Dab2, PAR-3, Gαi1 and Gαi3 associations are shown. (D) The expressions of listed proteins in WT and DKO MEFs, treated with/out VEGF (“-A”, 25 ng/mL) for 5 min, were tested by Western blotting assay. (E) Stable WT-MEFs with empty vector (“Vec”) or dominant negative Gαi3 (G202T) (“DN-Gαi3”) were treated with VEGF (“-A”, 25 ng/mL) for 5 min; associations between VEGFR2, Ephrin-B2, Dab2, PAR-3 and Gαi3 were tested by co-immunoprecipitation (“IP”) assay. (F) The expressions of listed proteins in total cell lysates were also tested.* P < 0.05 vs. WT MEFs in (C). # P < 0.05 vs. untreated control (“C”) cells in (D). # P < 0.05 vs. “Vec” in (E-F).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation, Dominant Negative Mutation, Control

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: shRNA-mediated knockdown of Gαi1/3 inhibits VEGF-induced VEGFR2 endocytosis and downstream signaling activation in HUVECs. (A-B) Stable HUVECs, expressing scramble control shRNA (“scr-shRNA”), Gαi1 shRNA, Gαi3 shRNA or Gαi1 plus Gαi3 shRNA, were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of the listed proteins. (C) VEGFR2 associations with Ephrin-B2, Dab2 and PAR-3 as well as Gαi1 and Gαi3 were tested by co-immunoprecipitation assay. (D) VEGFR2 expression in cell plasma membrane and in total cell lysates, along with the VEGFR2 endocytosis complex protein in total cell lysates were also tested. (E) Stable HUVECs expressing WT-Gαi3 (Ad-Gαi3) or the empty vector (“pDC315”), were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of the listed proteins.* P < 0.05 vs. HUVECs with “scr-shRNA” (B-C). # P < 0.05in (B). # P < 0.05 vs. “C” in (D). # P < 0.05 in (E).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, Knockdown, Activation Assay, Expressing, Control, Western Blot, Co-Immunoprecipitation Assay, Clinical Proteomics, Membrane, Plasmid Preparation

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: shRNA-mediated knockdown of Gαi1/3 inhibits VEGF-induced proliferation, invasion, migration and vessel-like tube formation in HUVECs. Stable HUVECs, expressing scramble control shRNA (“scr-shRNA”) or lentiviral Gαi1 plus Gαi3 shRNA, were treated with/without VEGF (“-A”, 10/25 ng/mL) for indicated times. Cell proliferation was tested by BrdU ELISA assay (A) and MTT OD assay (B) . Cell invasion and migration were tested by Transwell assay (C-D) and scratch wound healing assay (E-F) , respectively. HUVEC vessel-like tube formation was tested by the Matrigel-based capillary-genesis assay (G-H) . For the Transwell assay, ten random views of each condition were included to calculate the average number of invaded cells. For the scratch wound healing assay assay, ten random views of each condition were included to calculate the average migration distance. For the vessel-like tube formation assay, 25 random views were included to calculate the average number of formed vessels. # P < 0.05. Bar=100 μm.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, Knockdown, Migration, Expressing, Control, Enzyme-linked Immunosorbent Assay, Transwell Assay, Wound Healing Assay, Tube Formation Assay

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 shRNA inhibits in vivo angiogenesis. Eight-week-old male mice who had received a corneal alkali burn were treated with Gαi1 and Gαi3 shRNA lentivirus or scramble control shRNA (scr-shRNA). On day 7, corneal neovascularization (CoNV) was observed by slit-lamp ( A, bar=1 mm ) , and neovascularization was quantified (B) . (C-D) Mice injected with Gαi1 and Gαi3 shRNA lentivirus at P12 in the OIR model following removal from a 75% oxygen chamber had a significantly increased retinal vaso-obliteration ratio (VO%) on P17 than littermates injected with scramble control shRNA (scr-shRNA) (Bar=500 μm). Gαi1 and Gαi3 shRNA injection inhibited normal retinal vascular development at P17 as well. (E-F) Expressions of listed proteins in the retinal tissues of listed groups are shown. “No shRNA” stands for no shRNA lentivirus injected. # P < 0.05 in (E-F).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, In Vivo, Control, Injection

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: In vivo angiogenesis is impaired in Gαi1 and Gαi3 DKO mice. Eight-week-old wild-type (“WT”) and Gαi1 and Gαi3 double knockout (“DKO”) mice received a corneal alkali burn. On day 7, corneal neovascularization (CoNV) was observed by slit-lamp ( A, bar=1 mm ) , and neovascularization was quantified (B) . (C-D) Mice were subjected to OIR; representative images are shown at P12/P17, and retinal vaso-obliteration ratios (VO%) were quantified. Normal retinal physiological angiogenesis was inhibited in the DKO mice at P12 and P17 (Bar=500 μm). (E-F) Expressions of listed proteins in the retinal tissues of listed groups are shown. # P < 0.05.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: In Vivo, Double Knockout

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 upregulation in proliferative retinal tissues of PDR patients. The proliferative retinal membrane of six proliferative diabetic retinopathy (PDR) patients and retinas of three age-matched traumatic retinectomy patients were homogenized, and lysates were tested by Western blotting assay of the listed proteins (A-B) and Gαi/2/3 mRNAs ( A , right panel). Expressions of the listed proteins were quantified using ImageJ software.* P < 0.05.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Membrane, Western Blot, Software

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 1: LPA induced FOXM1 up-regulation in EOC cells. A. OVCA433 cells were starved for 16 hr prior to LPA (10 μM) treatment for different times as indicated. B. OVCA433 cells were starved for 16 hr prior to LPA treatment (6 hr) with different concentrations as indicated. The bar figures on the right panels are the summary of three independent experiments. C. OVCA433 cells were transfected with siRNA against YAP. The down-regulation of YAP and the effect on LPA-induced FOXM1 are shown. The antibody used to detect FOXM1 was from Sigma (Cat. Log # AV39518). *P < 0.05; **P < 0.01 and ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: Transfection

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 2: LPA1–3 and both of the PI3K and Rho pathways were involved in LPA-induced FOXM1. A. Pre-starved OVCA433 cells were treated with Ki16425 (10 μM) or Y27632 (10 μM) for 1 hr prior to LPA treatment (10 μM, 6 hr). FOXM1 was analyzed by Western blot. B. LPA (10 μM, 6 hr)-induced FOXM1 up-regulation were determined in LPA receptor specific siRNA-treated cells (48 hr post-transfection). C. Pre-starved OVCA433 cells were pretreated with PTX (100 ng/mL) for 16 hr; LY294002 (10 μM), U0126 (10 μM), or MK2203 (1 μM) for 1 hr, before LPA treatment for (10 μM, 6 hr). D. OVCA433 cells were starved overnight (16 hr), pretreated by OA(100 nM), C3(1 μg/mL), H89 (10 μM) for 1 hr, before LPA treatment for (10 μM, 6 hr). FOXM1 was detected using the Sigma antibody (Cat. Log # AV39518). The experiments were repeated at least three times. E and F. OVCA433 cells were transfected with dominant negative plasmids for 48 hr, starved and then treated with LPA (10 μM for 6 hr). Cell lysates were analyzed by Western blot. Representative results are shown. *P < 0.05; **P < 0.01; and ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: Western Blot, Transfection, Dominant Negative Mutation

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 3: LPA-transcriptionally regulated FOXM1B in OVCA433 cells. A. The transcriptional inhibitor ActD (1 μg/mL, 1 hr pre-treatment) and the translational inhibitor CHX (20 μg/mL, 1 hr pre-treatment) inhibited LPA-induced FOXM1 up-regulation by LPA (10 μM, 6 hr). FOXM1 was detected using the Sigma antibody (Cat. Log # AV39518). B. The mRNA levels of FOXM1 isoforms in OVCA433 cells with or without LPA (10 μM, 6 hr) treatment. FOXM1A was not detectible after 45 cycles of PCR amplification. Real-time RT-PCR conditions were described in Methods. *P < 0.05; **P < 0.01; and ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: Amplification, Quantitative RT-PCR

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 4: FOXM1 was functionally involved in cell proliferation, migration, and invasion in EOC cells. OVCA433 cells were starved and pretreated with different doses of thiostrepton (a selective inhibitor of FOXM1) for 1 hr prior to LPA treatment (10 μM, 6 hr). Thiostrepton dose-dependently reduced LPA-induced FOXM1 expression A. and inhibited LPA-induced cell migration and invasion in OVCA433 cells B. FOXM1 down-regulation by shRNA C. reduced cell migration and invasion induced by LPA and cell proliferation induced by 2% FBS D. The results are from three independent experiments. FOXM1 was detected using the Sigma antibody (Cat. Log# AV39518). *P < 0.05; **P < 0.01; and ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: Migration, Expressing, shRNA

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 5: LPA-induced FOXM1 in other EOC cell lines and FOXM1C was the dominant form in CAOV3 cells. A. LPA (10 μM and 6 hr) induced FOXM1 up-regulation in CAOV3 and OVCAR5 cells. CAOV3 and OVCAR5 cells expressed the FOXM1B with apparent MW of 80 KDa (Sigma, Cat. Log # AV39518). B. OVCA433 cells did not express FOXM1C, while CAOV3 cells expressed the FOXM1C with apparent MW of 100 KDa (Cell Signaling, Cat. Log # D12D5). C. CAOV3-FOXM1-KD cell line was established using shRNA against FOXM1 (detailed in Methods). The parental or control-shRNA transfected CAOV3 cells responded to LPA (10 μM, 6 hr) for FOXM1 up-regulation, which was blocked by KD-FOXM1. D. The dominant splicing form of FOXM1 in CAOV3 was FOXM1C. E. Comparison of the relative mRNA expression levels FOXM1 isoform in three EOC cell lines. F. KD-YAP and PTX inhibited LPA- induced FOXM1 up-regulation in CAOV3 cells. G. KD-FOXM1 inhibited LPA-induced cell migration and invasion. H. KD-FOXM1 inhibited FBS (2%)-induced cell proliferation. *P < 0.05; **P < 0.01; and ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: shRNA, Control, Transfection, Comparison, Expressing, Migration

Journal: Oncotarget

Article Title: FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

doi: 10.18632/oncotarget.4280

Figure Lengend Snippet: Figure 6: FOXM1 was functionally involved in tumorigenesis in vivo. A. Representative pictures of the tumors formed in Ctrl or FOXM1-KD cells injected mice. 7–10 weeks old female NOD/SCID mice were i.p. injected with 5 × 106 CAOV3 cells. Mice were euthanized 30 days after tumor cell injections and tumor development was analyzed. B. Tumor numbers and sizes were measured and compared. C. Effect of FOXM1-KD on CAOV3 generated ovarian tumor in NOD/SCID mice. D. FOXM1 related target genes were down- regulated at the mRNA level in tumors from CAOV3-KD injected mice, when compared to those in tumors from control cells. E. Human AREG and VEGF were reduced in ascites, measured by ELISA F. Human AREG and VEGF were reduced in serum, measured by ELISA *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Establish stable clones and drug selection concentrations FOXM1 shRNA lentiVirus vectors with GFP (OriGene, Rockville, MD) were transfected to 293T cells for virus packaging.

Techniques: In Vivo, Injection, Generated, Control, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 are required for VEGF-induced Akt-mTORC1 and Erk activation in mouse embryonic fibroblasts. (A-B) WT MEFs, Gαi1 or Gαi3 single knockout (SKO) MEFs, Gαi2 SKO MEFs, and Gαi1/3 double knockout (DKO) MEFs were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of listed proteins. (C-D) DKO MEFs were transiently transfected with the vector encoding Ad-Gαi1, Ad-Gαi3 or empty vector (pDC315); VEGF (“-A”, 25 ng/mL)-induced signaling was tested. The same sets of lysate samples were run in sister gels to test different proteins (same for all figures). Indicated protein was quantified via ImageJ software (same for all figures). “n=5” means quantification from five replicate blot data (same for all figures). Bars stand for mean ± SD (same for all Figures).* P < 0.05 vs. WT MEFs. # P < 0.05 in (A). # P < 0.05 vs. Vector MEFs in (D).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Activation Assay, Knock-Out, Double Knockout, Western Blot, Transfection, Plasmid Preparation, Software

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 are required for VEGF-induced VEGFR2 endocytosis in mouse embryonic fibroblasts. (A-B) WT MEFs were treated with VEGF (“-A”, 25 ng/mL) for indicated times; associations between VEGFR2, p-VEGFR2, Ephrin-B2, Dab2, PAR-3, Gαi1 and Gαi3 were tested by co-immunoprecipitation (“IP”) assay. (C) WT and DKO MEFs were treated with VEGF (“-A”, 25 ng/mL) for 5 min;VEGFR2, p-VEGFR2, Ephrin-B2, Dab2, PAR-3, Gαi1 and Gαi3 associations are shown. (D) The expressions of listed proteins in WT and DKO MEFs, treated with/out VEGF (“-A”, 25 ng/mL) for 5 min, were tested by Western blotting assay. (E) Stable WT-MEFs with empty vector (“Vec”) or dominant negative Gαi3 (G202T) (“DN-Gαi3”) were treated with VEGF (“-A”, 25 ng/mL) for 5 min; associations between VEGFR2, Ephrin-B2, Dab2, PAR-3 and Gαi3 were tested by co-immunoprecipitation (“IP”) assay. (F) The expressions of listed proteins in total cell lysates were also tested.* P < 0.05 vs. WT MEFs in (C). # P < 0.05 vs. untreated control (“C”) cells in (D). # P < 0.05 vs. “Vec” in (E-F).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Immunoprecipitation, Western Blot, Plasmid Preparation, Dominant Negative Mutation, Control

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: shRNA-mediated knockdown of Gαi1/3 inhibits VEGF-induced VEGFR2 endocytosis and downstream signaling activation in HUVECs. (A-B) Stable HUVECs, expressing scramble control shRNA (“scr-shRNA”), Gαi1 shRNA, Gαi3 shRNA or Gαi1 plus Gαi3 shRNA, were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of the listed proteins. (C) VEGFR2 associations with Ephrin-B2, Dab2 and PAR-3 as well as Gαi1 and Gαi3 were tested by co-immunoprecipitation assay. (D) VEGFR2 expression in cell plasma membrane and in total cell lysates, along with the VEGFR2 endocytosis complex protein in total cell lysates were also tested. (E) Stable HUVECs expressing WT-Gαi3 (Ad-Gαi3) or the empty vector (“pDC315”), were treated with VEGF (“-A”, 25 ng/mL) for indicated times, and were tested by Western blotting assay of the listed proteins.* P < 0.05 vs. HUVECs with “scr-shRNA” (B-C). # P < 0.05in (B). # P < 0.05 vs. “C” in (D). # P < 0.05 in (E).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, Knockdown, Activation Assay, Expressing, Control, Western Blot, Co-Immunoprecipitation Assay, Clinical Proteomics, Membrane, Plasmid Preparation

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: shRNA-mediated knockdown of Gαi1/3 inhibits VEGF-induced proliferation, invasion, migration and vessel-like tube formation in HUVECs. Stable HUVECs, expressing scramble control shRNA (“scr-shRNA”) or lentiviral Gαi1 plus Gαi3 shRNA, were treated with/without VEGF (“-A”, 10/25 ng/mL) for indicated times. Cell proliferation was tested by BrdU ELISA assay (A) and MTT OD assay (B) . Cell invasion and migration were tested by Transwell assay (C-D) and scratch wound healing assay (E-F) , respectively. HUVEC vessel-like tube formation was tested by the Matrigel-based capillary-genesis assay (G-H) . For the Transwell assay, ten random views of each condition were included to calculate the average number of invaded cells. For the scratch wound healing assay assay, ten random views of each condition were included to calculate the average migration distance. For the vessel-like tube formation assay, 25 random views were included to calculate the average number of formed vessels. # P < 0.05. Bar=100 μm.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, Knockdown, Migration, Expressing, Control, Enzyme-linked Immunosorbent Assay, Transwell Assay, Wound Healing Assay, Tube Formation Assay

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 shRNA inhibits in vivo angiogenesis. Eight-week-old male mice who had received a corneal alkali burn were treated with Gαi1 and Gαi3 shRNA lentivirus or scramble control shRNA (scr-shRNA). On day 7, corneal neovascularization (CoNV) was observed by slit-lamp ( A, bar=1 mm ) , and neovascularization was quantified (B) . (C-D) Mice injected with Gαi1 and Gαi3 shRNA lentivirus at P12 in the OIR model following removal from a 75% oxygen chamber had a significantly increased retinal vaso-obliteration ratio (VO%) on P17 than littermates injected with scramble control shRNA (scr-shRNA) (Bar=500 μm). Gαi1 and Gαi3 shRNA injection inhibited normal retinal vascular development at P17 as well. (E-F) Expressions of listed proteins in the retinal tissues of listed groups are shown. “No shRNA” stands for no shRNA lentivirus injected. # P < 0.05 in (E-F).

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: shRNA, In Vivo, Control, Injection

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: In vivo angiogenesis is impaired in Gαi1 and Gαi3 DKO mice. Eight-week-old wild-type (“WT”) and Gαi1 and Gαi3 double knockout (“DKO”) mice received a corneal alkali burn. On day 7, corneal neovascularization (CoNV) was observed by slit-lamp ( A, bar=1 mm ) , and neovascularization was quantified (B) . (C-D) Mice were subjected to OIR; representative images are shown at P12/P17, and retinal vaso-obliteration ratios (VO%) were quantified. Normal retinal physiological angiogenesis was inhibited in the DKO mice at P12 and P17 (Bar=500 μm). (E-F) Expressions of listed proteins in the retinal tissues of listed groups are shown. # P < 0.05.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: In Vivo, Double Knockout

Journal: Theranostics

Article Title: Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

doi: 10.7150/thno.26203

Figure Lengend Snippet: Gαi1 and Gαi3 upregulation in proliferative retinal tissues of PDR patients. The proliferative retinal membrane of six proliferative diabetic retinopathy (PDR) patients and retinas of three age-matched traumatic retinectomy patients were homogenized, and lysates were tested by Western blotting assay of the listed proteins (A-B) and Gαi/2/3 mRNAs ( A , right panel). Expressions of the listed proteins were quantified using ImageJ software.* P < 0.05.

Article Snippet: Lentivirus (20 μL virus per mL medium) containing shRNA of human Gαi1 (sc-105382-V) (Santa Cruz, CA) and/or human Gαi3 (sc-29325-V) (Santa Cruz, CA) was added.

Techniques: Membrane, Western Blot, Software

Journal: Infection and Immunity

Article Title: Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR–IκB Kinase–NF-κB Pathways in Dendritic Cells

doi: 10.1128/IAI.00190-16

Figure Lengend Snippet: Effects of NF-κB suppression on HO-1 expression in DCs treated with crude OMVs. (A and B) Activation of NF-κB in DCs stimulated with crude OMVs. BM-derived DCs (A) and DC2.4 cells (B) were treated with crude OMVs (50 μg/ml) for the indicated times. NF-κB DNA binding activity was assessed by EMSA. Immunoblot results for concurrent phospho-IκBα and lamin B in nuclear extracts under the same conditions are provided beneath the EMSA panels. Results are representative of more than three independent experiments. (C) Supershift assays using nuclear extracts from BM-derived DCs treated with crude OMVs (50 μg/ml) for 1 h were performed using Abs against p50, p52, p65, c-Rel, and Rel B. +, positive control; −, no extracts; IκBα-AA, nuclear extracts obtained from lentivirus containing IκBα superrepressor-transfected DC2.4 cells stimulated with crude OMVs (50 μg/ml) for 1 h. (D) DC2.4 cells were transfected with either lentivirus containing IκBα-superrepressor (IκBα-AA) or control virus ( GFP ). Transfected cells were stimulated with crude OMVs (50 μg/ml) for 1 h. NF-κB binding activity was assayed by EMSA (top panel). Transfected or untransfected cells were treated with crude OMVs (50 μg/ml) for 12 h. Expression of HO-1 and actin proteins was analyzed by immunoblotting (bottom panels). Results are representative of more than three independent experiments. (E) Transfected DC2.4 cells were treated with crude OMVs (50 μg/ml) for the indicated periods of time. Levels of HO-1 mRNA were analyzed by quantitative RT-PCR using a standard RNA. Values are expressed as means ± SD ( n = 5). β-Actin mRNA levels in each group remained relatively constant throughout the same periods (∼ 10 6 transcripts/μg total RNA). *, P < 0.05, for results compared with those in untransfected cells treated with crude OMVs. (F) DC2.4 cells were transfected with NF-κB p65 -specific siRNA or a nonsilencing siRNA (NS-RNA) as a control for 48 h, after which cells were combined with crude OMVs (50 μg/ml) for 1 h. Nuclear extracts were analyzed by immunoblotting with the indicated Abs (top panels). Transfected cells were stimulated with crude OMVs (50 μg/ml) for 12 h. Expression of HO-1 and actin proteins was analyzed by immunoblotting (bottom panels). Results shown are representative of more than three independent experiments. (G) DC2.4 cells were transfected with IκBα-AA or control virus ( GFP ). Transfected cells were stimulated with crude OMVs (50 μg/ml) for 6 h, and immunofluorescence microscopy was performed. Cells were stained with anti-HO-1 Ab (green), anti-phospho-p65 Ab (red), and 4′,6′-diamidino-2-phenylindole (DAPI; blue, nucleus). Data are representative of at least five experiments.

Article Snippet: Lentiviral vectors containing an Nrf2 shRNA plasmid (mouse) or IKKβ shRNA plasmid (mouse) and control lentivirus were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Derivative Assay, Binding Assay, Activity Assay, Western Blot, Positive Control, Transfection, Control, Virus, Quantitative RT-PCR, Immunofluorescence, Microscopy, Staining

Journal: Infection and Immunity

Article Title: Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR–IκB Kinase–NF-κB Pathways in Dendritic Cells

doi: 10.1128/IAI.00190-16

Figure Lengend Snippet: Effects of AP-1 suppression on HO-1 expression in DCs treated with crude OMVs. (A) Activation of AP-1 in DCs stimulated with crude OMVs. BM-derived DCs were treated with OMVs (50 μg/ml) for the indicated times. AP-1 DNA binding activity was assessed by EMSA. Immunoblot results for concurrent phospho-c-Jun and lamin B in nuclear extracts under the same conditions are provided beneath the EMSA panel. Results are representative of more than three independent experiments. (B) Supershift assays were performed using Abs against c-Jun, c-Fos, JunB, JunD, and FosB in BM-derived DCs stimulated with crude OMVs (50 μg/ml) for 1 h. +, positive control; −, no extracts; dn-c-jun, nuclear extracts obtained from with lentivirus-dn– c-jun -transfected DC2.4 cells stimulated with crude OMVs (50 μg/ml) for 1 h. (C) DC2.4 cells were transfected with either lentivirus containing dominant negative c-jun plasmid (dn– c-jun ) or control virus ( GFP ). Transfected cells were stimulated with crude OMVs (50 μg/ml) for 1 h. AP-1 binding activity was assayed by EMSA. Transfected or untransfected cells were treated with OMVs (50 μg/ml) for 12 h (top panel). Expression of HO-1 and actin proteins was analyzed by immunoblotting (bottom panels). Results are representative of more than three independent experiments. (D) Transfected DC2.4 cells were treated with crude OMVs (50 μg/ml) for the indicated periods of time. Levels of HO-1 mRNA were analyzed by quantitative RT-PCR using a standard RNA. Values are expressed as means ± SD ( n = 5). β-Actin mRNA levels in each group remained relatively constant throughout the same periods (∼ 10 6 transcripts/μg total RNA). *, P < 0.05, for results compared with those in untransfected cells treated with crude OMVs. (E) DC2.4 cells were transfected with AP-1 c-jun -specific siRNA or nonsilencing siRNA (NS-RNA) as a control for 48 h, after which cells were combined with crude OMVs (50 μg/ml) for 1 h. Nuclear extracts were analyzed by immunoblotting with the indicated Abs (top panels). Transfected cells were stimulated with crude OMVs (50 μg/ml) for 12 h. Expression of HO-1 and actin proteins was analyzed by immunoblotting (bottom panels). Results shown are representative of more than three independent experiments. (F) Murine BM-derived DCs were preincubated with NF-κB inhibitor Bay 11-7082 (50 μM) or AP-1 inhibitor SR11302 (10 μM) for 30 min, followed by stimulation with crude OMVs (50 μg/ml) for an additional 12 h. Expression levels of HO-1 were measured by ELISA (means ± SEM; n = 5). *, P < 0.05, for results compared with those with OMV alone. NS, statistically nonsignificant.

Article Snippet: Lentiviral vectors containing an Nrf2 shRNA plasmid (mouse) or IKKβ shRNA plasmid (mouse) and control lentivirus were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Derivative Assay, Binding Assay, Activity Assay, Western Blot, Positive Control, Transfection, Dominant Negative Mutation, Plasmid Preparation, Control, Virus, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Developmental Cell

Article Title: Subcellular mRNA Localization Regulates Ribosome Biogenesis in Migrating Cells

doi: 10.1016/j.devcel.2020.10.006

Figure Lengend Snippet:

Article Snippet: MCP-EGFP expression plasmid, as well as the VSV and deltaR lentiviral packaging vectors were a gift from Carme Gallego (IBMB, Barcelona). rtTA-N144 ( ) was a gift from Andrew Yoo (Addgene plasmid # 66810). pTK-Twist & pTK-Snail lentiviral inducible expression plasmids ( ) were a gift from Bob Weinberg (Addgene plasmids #36977 & #36976).

Techniques: Virus, Cloning, Recombinant, Western Blot, Staining, Plasmid Preparation, RNAscope, Multiplex Assay, Control, CRISPR, Modification, Transfection, Fractionation, Peptide Fractionation, Sequencing, Cell Viability Assay, Viability Assay, RNA HS Assay, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Quantitative Proteomics, Multiplex sample analysis, Generated, Stable Transfection, Expressing, Negative Control, Software, Membrane, Cell Culture

Journal: Scientific Reports

Article Title: Rescuing Infertility of Pick1 Knockout Mice by Generating Testis-specific Transgenic Mice via Testicular Infection

doi: 10.1038/srep02842

Figure Lengend Snippet: (A) Schematic diagram of the pWPXL lentiviral vector. LTR: long terminal repeat. RRE, ref-responsive element. cPPT, central polypurine tract. pEF1α, human elongation factor alpha promoter; GFP, green fluorescent protein. WPRE, woodchuck hepatitis virus posttranscriptional control element. SIN, self-inactivating. (B) Schematic diagram of intratubular microinjection through the efferent duct. (C) Western blotting analysis of injected testes, 1 week after microinjection. GAPDH served as the loading control. Cell lysate from pWPXL-GFP lentivirus infected 293T cells was used as positive control. Full-length blots are presented in . (D) Cryosection analysis of the injected testis and un-injected control. Nucleus was labeled by DAPI. Cryosections were cut at 10 μm in thickness. Scale bar, 20 μm. (E) Western blotting analysis of injected testes, 2.5 months after microinjection. GAPDH served as the loading control. Full-length blots are presented in . (F) Cryosection analysis of the injected testis, 2.5 months after microinjection. Scale bar, 20 μm.

Article Snippet: The GFP-containing pWPXL lentiviral vector (pWPXL-GFP) was from Addgene (plasmid 12257, Didier Trono) .

Techniques: Plasmid Preparation, Virus, Control, Microinjection, Western Blot, Injection, Infection, Positive Control, Labeling

Journal: Scientific Reports

Article Title: Rescuing Infertility of Pick1 Knockout Mice by Generating Testis-specific Transgenic Mice via Testicular Infection

doi: 10.1038/srep02842

Figure Lengend Snippet: Transgenic mice from seminiferous tubule microinjection of lentivirus

Article Snippet: The GFP-containing pWPXL lentiviral vector (pWPXL-GFP) was from Addgene (plasmid 12257, Didier Trono) .

Techniques: Transgenic Assay, Microinjection, Injection

Journal: Scientific Reports

Article Title: Rescuing Infertility of Pick1 Knockout Mice by Generating Testis-specific Transgenic Mice via Testicular Infection

doi: 10.1038/srep02842

Figure Lengend Snippet: (A) Schematic diagram of the pWPXL-based constructs. IRES, internal ribosome entry site. pCMV, human cytomegalovirus immediate early promoter. Arrows indicate the design of exogenous-PICK1 genotyping (GT-Exo-PICK1) PCR primers. (B) Western blotting analysis of the HEK293T cells transfected with different lentiviral vectors. (C) Western blotting analysis of the testes from the injected Pick1 knockout mice. The sample from wild-type mouse was used as the positive control for PICK1 and negative control for GFP. (D) Western blotting analysis of the expression of GFP and PICK1 in the Pick1&Gfp dual-gene transgenic mice. KO, Pick1 knockout sample, was used as a negative control for PICK1. G554M, the Gfp transgenic male mouse, was used as a positive control for GFP. (E) Western blotting analysis of the tissue distribution of GFP expression in the transgenic mice. Full-length blots are presented in .

Article Snippet: The GFP-containing pWPXL lentiviral vector (pWPXL-GFP) was from Addgene (plasmid 12257, Didier Trono) .

Techniques: Construct, Western Blot, Transfection, Injection, Knock-Out, Positive Control, Negative Control, Expressing, Transgenic Assay