Journal: Gene therapy

Article Title: The use of polyethylenimine-DNA to topically deliver hTERT to promote hair growth.

doi: 10.1038/gt.2011.62

Figure Lengend Snippet: Figure 1 Flow cytometric analysis and fluorescence detection of sorted cells double stained for integrin b1 and CD 71. The integrin b1CD71 (a) and integrin b1+CD71 (b) cells were collected by FACS and examined by flow cytometry, respectively. Integrin b1CD71 cells are shown in the lower left-hand quadrant of each contour plot, whereas integrin b1+CD71 cells are shown in the lower right-hand quadrant, with percentage of each sorted cell population. Photo images of integrin b1CD71 (c) and integrin b1+CD71 cells (d). Integrin b1 was obvious by green fluorescence and CD71 was obvious by red fluorescence. Nuclei were counterlabeled with 4,6-diamidino-2-phenylindole (DAPI) (scale bar¼10 mm). The color reproduction of this figure is available on the html full text version of the manuscript.

Article Snippet: Stem cells, which expressed integrin a6, were detected by staining with anti-integrin a6 mouse monoclonal antibody (sc-59920; Santa Cruz Biotechnology, Inc.) followed by anti-mouse secondary antibody conjugated with Alexa Fluor 647 (A21235; Invitrogen) and with anti-hTERT with anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate.

Techniques: Staining, Cytometry

Journal: Gene therapy

Article Title: The use of polyethylenimine-DNA to topically deliver hTERT to promote hair growth.

doi: 10.1038/gt.2011.62

Figure Lengend Snippet: Figure 3 hTERT expression in DNA–PEI complex transfected b1+CD71 keratinocytes increased expression of cell proliferation marker. (a–d) Detection of hTERT expression was detected by immunofluorescent staining (Allophycocyanin, APC) in integrin b1+CD71 keratinocytes after (a) treatment of water (Neg), (b) naked pLC–hTERT plasmid (DNA), (c) PEI vector only (PEI) and (d) PEI–pLC–hTERT complex at N/P¼7 (N/P). Panels (e–h) showed that immunofluorescent staining of PCNA (Allophycocyanin, APC) after treatment of water (e), DNA (f), PEI (g) and hTERT–DNA–PEI complex (h). Sections stained with antibodies that were color-coded according to the fluorescent tags of the secondary antibodies. Western blot analyses of hTERT (i) and PCNA (j) expression in cells transfected with the different treatments indicated on the top (Neg, water; DNA, pLC–hTERT plasmid; P7, PEI at molar ratio¼7; P15, PEI at molar ratio¼15; N/P7, hTERT–DNA–PEI complex at N/P ratio¼7; N/P15, hTERT–DNA–PEI complex at N/P ratio¼15). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for normalization of the total protein amounts. The color reproduction of this figure is available on the html full text version of the manuscript.

Article Snippet: Stem cells, which expressed integrin a6, were detected by staining with anti-integrin a6 mouse monoclonal antibody (sc-59920; Santa Cruz Biotechnology, Inc.) followed by anti-mouse secondary antibody conjugated with Alexa Fluor 647 (A21235; Invitrogen) and with anti-hTERT with anti-rabbit secondary antibody conjugated with fluorescein isothiocyanate.

Techniques: Expressing, Transfection, Marker, Staining, Plasmid Preparation, Western Blot, Control

Journal: Scientific reports

Article Title: Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

doi: 10.1038/s41598-024-51521-w

Figure Lengend Snippet: Figure 2. Identification of importin α subtypes interacting with IL-1α. The isolation of His-tag-fused IL-1α via IMAC was conducted using cell lysates from HeLa cells expressing His-tag-fused IL-1α. (a) Immunoblotting was performed using anti-His and anti-GAPDH antibodies; FT represents the flow-through fraction, and Elution represents the imidazole elution fraction. (b) anti-HAX-1, anti-importin α1, anti-importin α3, anti-importin α4, anti-importin α5, anti-importin α6, and anti-importin α7 antibodies were used for immunoblotting. Arrowheads indicate bands corresponding to the expected molecular weight of each protein. We confirmed the reproducibility of the results (Supplementary Fig. S1). The full-length blots are shown in Supplementary Information, Fig. 2.

Article Snippet: Monoclonal mouse anti-IL-1α (sc-271618), anti-GAPDH (sc-47724), anti-importin α1 (sc-55538), anti-importin α4 (sc-514101), anti-importin α5 (sc-101292), anti-importin α7 (sc-390055) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Isolation, Expressing, Western Blot, Molecular Weight

Journal: Scientific reports

Article Title: Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

doi: 10.1038/s41598-024-51521-w

Figure Lengend Snippet: Figure 3. Analysis of the effect of importin α1 on IL-1α expression and nuclear translocation. (a) Cell lysates from HeLa cells transfected with 5 nM negative-control siRNA or 2.5 or 5 nM importin α1 siRNA and then transfected 24 h later with His-tag-fused IL-1α were subjected to SDS-PAGE and immunoblotting with anti- importin α1, anti-His, and anti-β-actin antibodies. (b) The nuclear and cytoplasmic fractions of HeLa cells transfected with importin α1 siRNA or negative-control siRNA and His-tag-fused IL-1α were obtained, and each fraction was then subjected to immunoblotting using anti-importin α1, anti-His, anti-lamin B1, and anti- β-actin antibodies. (c) IL-1α protein levels were measured in the nuclear and cytoplasmic fractions. Results are expressed as the nuclear/cytoplasmic ratio. Data are presented as the mean ± SD. Significant difference (P < 0.05) based on an unpaired Student’s t-test is indicated by asterisk (n ≥ 3). The full-length blots are shown in Supplementary Information, Fig. 3.

Article Snippet: Monoclonal mouse anti-IL-1α (sc-271618), anti-GAPDH (sc-47724), anti-importin α1 (sc-55538), anti-importin α4 (sc-514101), anti-importin α5 (sc-101292), anti-importin α7 (sc-390055) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Translocation Assay, Transfection, Negative Control, SDS Page, Western Blot

Journal: Scientific reports

Article Title: Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

doi: 10.1038/s41598-024-51521-w

Figure Lengend Snippet: Figure 4. Analysis of the behavior of importin β1 in IL-1α nuclear translocation. (a) After isolation of His- tag-fused protein via IMAC from HeLa cells expressing His-tag-fused IL-1α, the obtained fractions during IMAC were analyzed using Western blotting with anti-His, anti-importin β1, and anti-GAPDH antibodies. Input lysate of HeLa cells before IMAC, FT flow-through fraction, Elution imidazole elution fraction. (b) Coimmunoprecipitation was performed with an anti-importin β1 antibody using HeLa cells expressing His-tag- fused IL-1α, and the input, supernatant fraction, and pellet fraction were analyzed by Western blotting using anti-importin β1, anti-importin α1, anti-p65, anti-His, and anti-GAPDH antibodies. Input lysate of HeLa cells before immunoprecipitation, Sup supernatant fraction, Pellet pellet fraction. The full-length blots are shown in Supplementary Information, Fig. 4.

Article Snippet: Monoclonal mouse anti-IL-1α (sc-271618), anti-GAPDH (sc-47724), anti-importin α1 (sc-55538), anti-importin α4 (sc-514101), anti-importin α5 (sc-101292), anti-importin α7 (sc-390055) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay, Isolation, Expressing, Western Blot, Immunoprecipitation

Journal: Scientific reports

Article Title: Analysis of the effects of importin α1 on the nuclear translocation of IL-1α in HeLa cells.

doi: 10.1038/s41598-024-51521-w

Figure Lengend Snippet: Figure 5. Components of complexes for nuclear translocation of IL-1α. Left side: general transport complex of substrate containing cNLS, which forms a complex with importin α, importin β1, and substrate transported to the nucleus in an importin β1-dependent manner. Right side: the IL-1α binds to importin α1 and an unknown protein, and is subsequently transported into the nucleus independently of importin β1.

Article Snippet: Monoclonal mouse anti-IL-1α (sc-271618), anti-GAPDH (sc-47724), anti-importin α1 (sc-55538), anti-importin α4 (sc-514101), anti-importin α5 (sc-101292), anti-importin α7 (sc-390055) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: Upregulation of Nav1.1 and downregulations of KCa3.1 and α 6 subunit of GABA A receptors in the cortex from Il-4 −/− mice and supplemental IL-4 increases KCa3.1 and α 6 mRNA expressions. Upregulation of Nav1.1 mRNA expression and downregulations of KCa3.1 and α 6 subunit of GABA A receptors mRNA expression, in cortical tissues (A) and cortical neurons (B) from Il-4 −/− mice. (C) Nav1.1 protein expression in primary mouse cortical neurons by immunostaining and upregulation of Nav1.1 protein in Il-4 −/− mice ( n = 6 mice). (D) The image staining with KCa3.1 antibody (green), NeuN antibody (red, a neuronal-specific nucleus marker) and DAPI (blue, a nucleus marker). Downregulation of KCa3.1 protein in Il-4 −/− mice ( n = 4 mice, Mann Whitney test). (E) Downregulation of α 6 subunit of GABA A protein in Il-4 −/− mice ( n = 4 mice). Increased mRNA expressions of KCa3.1 and α 6 subunit in Il-4 −/− (F) and Il-4 +/+ (G) cortical neurons after supplementing IL-4 (20 ng/mL) in culture for 7 days. Data are expressed as the mean ± SEM, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with their controls. The numbers at the bottom of the bars indicate the number of repeats or mice in the group.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Expressing, Immunostaining, Staining, Marker, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Deficiency of anti-inflammatory cytokine IL-4 leads to neural hyperexcitability and aggravates cerebral ischemia–reperfusion injury

doi: 10.1016/j.apsb.2020.05.002

Figure Lengend Snippet: A proposed molecular mechanism underlying increased neural excitabilities and susceptibility to ischemic injury caused by IL-4 deficiency. IL-4 binding to IL-4R actives IL-4 pathway. IL-4 deficiency alters gene transcriptions by downregulating the Kcnn4 gene encoding KCa3.1 protein and Gabra6 gene encoding GABA A receptor chloride channel and upregulating the Scna1 gene encoding Nav1.1 protein through IL-4 signaling pathways. Downregulation of KCa3.1 channels and tonic GABA A receptors can reduce potassium outflow and chloride inflow in neurons, leading to enhanced neuronal firings through membrane depolarization. The upregulation of Nav1.1 channels can increase sodium inflow in neurons. All these alterations can enhance neuronal hyperexcitability and glutamate release from excitatory axon terminals, ultimately increasing susceptibility to ischemic injury. Conversely, enhancement of IL-4 signaling through supplemental IL-4 can increase KCa3.1 and α 6 subunit of GABA A receptors in cortical neurons and reverse neuronal hyperexcitability, thus exerting neuroprotection against ischemic injury.

Article Snippet: After blocking by 5% milk, PVDF membranes were incubated with primary antibodies at 4 °C overnight such as rat anti-IL-4 antibody (1:500, Abcam, ab11524), mouse monoclonal anti-KCa3.1 antibody (1:250, Alomone, ALM-051), rabbit anti-Nav1.1 antibody (1:250, Alomone, ASC-001), rabbit GABA A R α 6 Polyclonal Antibody (1:200, Alomone, AGA-004), mouse anti- β -actin antibody (1:5000, Abcam) and mouse anti-GAPDH antibody (1:5000, Abcam).

Techniques: Binding Assay, Protein-Protein interactions, Membrane, Patch Clamp, Immunofluorescence

Journal: Cell reports

Article Title: Cerebellum-Specific Deletion of the GABA A Receptor δ Subunit Leads to Sex-Specific Disruption of Behavior

doi: 10.1016/j.celrep.2020.108338

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-GABA A receptor-α6 , GeneTex , Cat# GTX130947.

Techniques: Recombinant, RNAscope, Knock-Out, Software

Journal: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Evaluation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A western blot of α6 nAChR subunit expression and antigen block in bilateral ventral tegmental tissue punches collected from male Sprague Dawley rats. n=2 animals total. GAPDH is used as a loading control.

Article Snippet: Thus, the purpose of this study is to validate the specificity of the commercially available polyclonal α6 nAChR subunit antibody from Alomone Labs (cat. #: ANC-006, Jerusalem, Israel).

Techniques: Western Blot, Expressing, Blocking Assay

Journal: Psychopharmacology

Article Title: Specificity of a rodent alpha(α)6 nicotinic acetylcholine receptor subunit antibody

doi: 10.1007/s00213-019-05413-x

Figure Lengend Snippet: Genetic validation of an alpha(α)6 nicotinic acetylcholine receptor subunit antibody. A.) Western blot and B.) relative band intensity quantification of α6 nAChR subunit expression and antigen block from whole brain tissue collected from male and female WT and α6 KO C57BL/6J mice, n=4 animals/genotype; **p< 0.01 and ***p< 0.001 α6 antibody vs. α6 antibody + antigen normalized signal. GAPDH is used as a loading control.

Article Snippet: Thus, the purpose of this study is to validate the specificity of the commercially available polyclonal α6 nAChR subunit antibody from Alomone Labs (cat. #: ANC-006, Jerusalem, Israel).

Techniques: Western Blot, Expressing, Blocking Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: S100P is expressed in trophoblasts including extravillous trophoblasts and more significantly during the first trimester in placental tissues. Expression of S100P proteins was analysed on both lysates ( A , B ) and sections ( C , E ) obtained from different paraffin-embedded placental samples from different gestational periods (first trimester (n = 5), second trimester (n = 4), or third trimester (n = 7). Proteins extracted from paraffin-embedded placental block sections (1 st trimester; 2 nd trimester and 3 rd trimester) at equal loading were separated by SDS-PAGE electrophoresis. Western blotting was carried out and membranes probed for S100P or α-tubulin and cropped images are presented ( A ). Levels of S100P were measured by densitometry analysis after Western blotting and normalised to α-tubulin for all samples (first trimester (n = 5), second trimester (n = 4), or third trimester (n = 7). Data is presented as percentage means ± SD of 2 independent experiments compared to the first trimester ( B ). Immunohistochemistry staining using a goat polyclonal S100P antibody and counterstaining on human placental tissues was performed as described in Methods. Arrows indicate cytotrophoblast cells (CT), syncytium (ST). Stroma is also highlighted. Bar corresponds to 150 μm ( C ). Quantification of S100P DAB staining and intensity in 1 st , 2 nd and 3 rd trimester serial sections. Data of an individual representative experiment is presented as the mean values ± SD of 3 independent samples ( D ). Statistical analysis ( B , D ) show ± SD compared to the first trimester samples of an individual representative experiment. *P < 0.05 and **P < 0.01 (one way- ANOVA). Immunohistochemistry staining using a panel of trophoblast marker antibodies (cytokeratin 7 (CK7), HLA-G, integrin α6 (CD49F) and S100P antibody) and counterstaining on anchoring columns of serial human placental tissues was performed as described in Methods. Arrows indicate cytotrophoblast cells (CT), syncytium (ST). Stroma is also highlighted. Bar corresponds to 100 μm ( E ).

Article Snippet: Non-specific protein binding was blocked by incubation with either 1% (v/v) normal bovine serum and 5% (v/v) goat IgG (Vector Labs, UK) or 10% (v/v) normal swine serum (Vector Labs, UK) for 1 hour and sections were then incubated with a goat polyclonal anti-S100P antibody (R&D, UK), a rabbit monoclonal anti-S100P antibody (Abcam, UK), a mouse monoclonal anti-HLA-G antibody (Abcam, UK), a rabbit polyclonal anti-CD49f (integrin α6) antibody (Abcam, UK) or an mouse monoclonal anti cytokeratin7 antibody (Leica Biosystems, UK) at 4 °C overnight (Supplementary Table ).

Techniques: Expressing, Blocking Assay, SDS Page, Electrophoresis, Western Blot, Immunohistochemistry, Staining, Marker

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

doi: 10.1016/j.bbamcr.2016.10.012

Figure Lengend Snippet: Fig. 2. SALL4 up-regulates integrin α6 and β1 expression. (A) The scatter plot shows the gene expressions in the control and SALL4 knockdown SUM159 cells. Integrin genes reduced by SALL4 knockdown are indicated. The accession numbers of the shGFP and shSALL4 data are DRA004721 and DRA004722, respectively. (B) Relative expression levels of integrin family genes are shown (n = 3). In each gene, mean expression level of the shGFP cells was defined as 1. Arrowheads point the genes that had expression reduced to less than half of that of the controls by SALL4 knockdown in both SUM159 and MDA-MB-231 cells. (C) Images show cells with integrin α6 and β1 knockdown. (D) The results of the Boyden chamber assays are shown (n = 4). MDA-MB-231 cells were used. The vertical axis indicates the number of migrated cells. (E) Reporter assays using the integrin α6 and β1 gene promoters were performed (n = 4). The vertical axis indicates the relative light units of luciferase. The mean value of the FLAG control was defined as 1. (F) Enrichment of the integrin α6 and β1 promoter region is shown. MDA-MB-231 cells were used. Chromatin immunoprecipitation samples were used for PCR amplification with primers for the integrin α6 and β1 promoters. ChIP: chromatin immunoprecipitation. Scale bars indicate 100 μm. Student's t-test was used for statistical analyses. ⁎P b 0.05, ⁎⁎P b 0.01. Error bars represent the standard deviation.

Article Snippet: The primary antibodies were anti-phosphopaxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, USA, 1:20 dilution), anti-GM130 antibody (Cell Signaling Technology, 12480, 1:3200 dilution), anti-integrin α6 antibody (Acris Antibodies GmbH,Herford, Germany, 1:100dilution) and anti-integrinβ1 antibody (GeneTex, GTX23167, Alton Pkwy Irvine, CA, USA, 1:10 dilution).

Techniques: Expressing, Control, Knockdown, Luciferase, Chromatin Immunoprecipitation, Standard Deviation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

doi: 10.1016/j.bbamcr.2016.10.012

Figure Lengend Snippet: Fig. 3. SALL4-regulated integrin α6 and β1 expression is required for spindle-shaped morphology and cell migration in basal-like breast cancer cells. (A) Boyden chamber assays were performed with cells overexpressing integrin α6 and β1 (n = 4 in control, n = 5 in the α6 and β1 overexpressing groups). The vertical axis indicates the number of migrated cells. (B) Cells overexpressing integrin α6 and β1 are shown. (C) Images of immunostaining with anti-phosphorylated paxillin are shown. Arrowheads indicate the immunoreaction signals. For simplicity, not all signals are pointed. (D) Graphs show the ratio of the FA-rich region. The cell numbers were 33 in control;shGFP, 36 in control;shSALL4, 45 in α6v1,β1;shGFP, 37 in α6v1,β1;shSALL4, 39 in α6v2,β1;shGFP and 33 in α6v2,β1;shSALL4. (E) Immunoblotting for FAK was performed. (F) The intensity of phosphorylated FAK and the total FAK bands were measured. Relative FAK phosphorylation levels to shGFP are graphed (n = 3). Scale bars indicate 100 μm in B, and 20 μm in C. Student's t-test was used. ⁎P b 0.05, ⁎⁎P b 0.01. Error bars represent the standard deviation.

Article Snippet: The primary antibodies were anti-phosphopaxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, USA, 1:20 dilution), anti-GM130 antibody (Cell Signaling Technology, 12480, 1:3200 dilution), anti-integrin α6 antibody (Acris Antibodies GmbH,Herford, Germany, 1:100dilution) and anti-integrinβ1 antibody (GeneTex, GTX23167, Alton Pkwy Irvine, CA, USA, 1:10 dilution).

Techniques: Expressing, Migration, Control, Immunostaining, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

doi: 10.1016/j.bbamcr.2016.10.012

Figure Lengend Snippet: Fig. 4. Integrin α6 and β1 form a heterodimer. (A) Co-immunoprecipitation samples were used for immunoblotting with an anti-integrin β1 antibody. IP: immunoprecipitation. IB: immunoblotting. (B) Confocal images of immunostained cells are shown. Antibodies for integrin α6 and β1 were used for double-immunostaining. (C-F) Binding assays were performed (n = 4). The number of cells bound to the BSA blocking solution and the recombinant laminin-511 E8 fragment are shown in C,E and D,F, respectively. The vertical axis indicates the number of bound cells. SUM159 and MDA-MB-231 cells were used in C,D. MDA-MB-231 cells overexpressing integrin α6 and β1 were used in E,F. Scale bars indicate 20 μm. Student's t-test was used. n.s.: not significant, ⁎⁎P b 0.01. Error bars represent the standard deviation.

Article Snippet: The primary antibodies were anti-phosphopaxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, USA, 1:20 dilution), anti-GM130 antibody (Cell Signaling Technology, 12480, 1:3200 dilution), anti-integrin α6 antibody (Acris Antibodies GmbH,Herford, Germany, 1:100dilution) and anti-integrinβ1 antibody (GeneTex, GTX23167, Alton Pkwy Irvine, CA, USA, 1:10 dilution).

Techniques: Immunoprecipitation, Western Blot, Double Immunostaining, Binding Assay, Blocking Assay, Recombinant, Standard Deviation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

doi: 10.1016/j.bbamcr.2016.10.012

Figure Lengend Snippet: Fig. 6. The SALL4 - integrin α6β1 network modulates Rho activity to promote cell migration. (A) RhoA protein level was not changed by SALL4 knockdown in MDA-MB-231 cells. (B) RhoA activity was measured with RhoA G-LISA Activation Assay Kit. Relative RhoA activity to shGFP was calculated (n = 3). (C) Immunofluorescence images of phosphorylated paxillin are shown. A ROCK inhibitor, Y-27632, was used. Arrowheads indicate the signals. For simplicity, not all signals are pointed. (D) The FA-rich region was analyzed. The cell numbers analyzed were 43 in SUM159;shGFP, 35 in SUM159;shSALL4 with water, 43 in SUM159;shSALL4 with the ROCK inhibitor, 41 in MDA-MB-231;shGFP, 36 in MDA-MB-231;shSALL4 with water and 31 in MDA-MB-231;shSALL4 with the ROCK inhibitor. (E) Boyden chamber assays were performed with the ROCK inhibitor (n = 4). The vertical axis indicates the number of migrated cells. Scale bars indicate 20 μm. Student's t-test was used. ⁎⁎P b 0.01. Error bars represent the standard deviation.

Article Snippet: The primary antibodies were anti-phosphopaxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, USA, 1:20 dilution), anti-GM130 antibody (Cell Signaling Technology, 12480, 1:3200 dilution), anti-integrin α6 antibody (Acris Antibodies GmbH,Herford, Germany, 1:100dilution) and anti-integrinβ1 antibody (GeneTex, GTX23167, Alton Pkwy Irvine, CA, USA, 1:10 dilution).

Techniques: Activity Assay, Migration, Knockdown, Activation Assay, Standard Deviation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: The Sal-like 4 - integrin α6β1 network promotes cell migration for metastasis via activation of focal adhesion dynamics in basal-like breast cancer cells.

doi: 10.1016/j.bbamcr.2016.10.012

Figure Lengend Snippet: Fig. 7. The SALL4 - integrin α6β1 network is required for in vivo cell migration. (A) Metastasis tracking assay of transplanted SUM159 cells in zebrafish larvae. In the control condition (shGFP), the SUM159 cells (magenta) moved to the anterior (yellow square) and posterior (blue square) from the injected site at day 3. The metastasized cells (arrowheads) were localized outside of the vasculatures (green, fli1-EGFP). On the other hand, the SALL4 knockdown cells did not migrate out from the abdominal cavity. Scale bars indicate 500 μm. (B,C) Statistical data for the metastasis assay. The ratio of fish with the metastasis was decreased under the shSALL4 condition in the both of SUM159 and MDA-MB-231 cells (B). The shSALL4-induced phenotype was rescued by overexpression of integrin α6 and β1 (C). Fisher's exact test was used. ⁎⁎P b 0.01.

Article Snippet: The primary antibodies were anti-phosphopaxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, USA, 1:20 dilution), anti-GM130 antibody (Cell Signaling Technology, 12480, 1:3200 dilution), anti-integrin α6 antibody (Acris Antibodies GmbH,Herford, Germany, 1:100dilution) and anti-integrinβ1 antibody (GeneTex, GTX23167, Alton Pkwy Irvine, CA, USA, 1:10 dilution).

Techniques: In Vivo, Migration, Control, Injection, Knockdown, Over Expression

Journal: Nature Communications

Article Title: Integrin α3β1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis

doi: 10.1038/s41467-022-31981-2

Figure Lengend Snippet: a , b Total RNA was extracted from 3 NECs (376, 422, and 623) and 3 TECs (isolates cw880, ccf2352, and ccf2390) when similar densities of tubes had formed on Matrigel, and c , d from monolayers of 6 TECs (cw850, cw880, ccf2277, ccf2352, ccf2388, and ccf2390) and 3 NECs (375, 422, and 623), followed by gene expression microarray (HG-U219). a , c Genes upregulated over 2-fold in TECs compared to NECs were subject to gene set enrichment and pathway analysis using DAVID Bioinformatics Resources 6.8 ( https://david.ncifcrf.gov ), where p -values were calculated by Fisher’s exact test. b , d Expression profiles of integrins and genes relevant to angiogenesis both on Matrigel and in monolayer are shown as heatmaps; p -values were obtained by two-sided exact-Wilcoxon-rank-sum tests, without adjustment for multiple comparisons. e Cell lysates derived from the same experiment with monolayers of NECs (623, 376, and 422) and TECs (ccf2445, ccf2566, and ccf2515) were subjected to western blot analysis with the indicated antibodies in parallel. f Representative confocal images of integrin α3 staining of tubes formed by NECs (376) or TECs (ccf2566) plated on Matrigel (24 h). Graph represents the integrated densities of integrin α3-immunofluorescence. g Paraffin sections of human GBM and normal brain (NB) samples were double-labeled for integrin α3 and vwf, followed by DAPI staining. Representative images shown. Colocalization of the integrin α3 signal with the vwf signal for GBM-sections was 86%, based on the Manders’ Coefficient (M1 = 0.863; M2 = 0.515). h Human GBM tissue arrays containing NB were double-labeled for vwf and integrin α3, CD151, or integrin α6. Statistical analysis: two-sided Wilcoxon-rank-sum-tests; red lines denote means and black lines denote 95% confidence intervals. i Tumor sections from mouse glioma were double-labeled by immunofluorescence for integrin α3, CD151, or integrin α6 and CD31. Average fluorescence intensities of integrin α3, CD151, or integrin α6 on CD31-positive pixels/field were obtained from tumor or adjacent NB using ImageJ and represented as boxplots. Boxes indicate first and third quartiles, bands indicate medians, and whiskers indicate ±1.5 interquartile range. Statistical analysis: linear mixed model; red bars, means. ( h , i ) On the x axis, n = number of different fields. e – g Independent experiments were performed at least two times with similar results. Source data are provided as a source file.

Article Snippet: For immunofluoresence analysis of mouse glioma, the primary antibodies used were rabbit anti-integrin α3 subunit (Millipore-Sigma, AB1920), rabbit anti-integrin α6 subunit (Bioss, BS-2641R), rabbit anti-CD151 (Epitomics, 5901-1, clone EP6875) and rat anti-mouse CD31 (Dianova, DIA-310) and the secondary antibodies were Alexa-Fluor-568-conjugated goat anti-rabbit IgG (A11036) and Alexa-Fluor-488-conjugated goat anti-rat IgG (A21208) from ThermoFisher Scientific.

Techniques: Expressing, Microarray, Derivative Assay, Western Blot, Staining, Immunofluorescence, Labeling, Fluorescence

Journal: Nature Communications

Article Title: Integrin α3β1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis

doi: 10.1038/s41467-022-31981-2

Figure Lengend Snippet: a , c NECs (623) or TECs (isolate ccf2445) were incubated with function blocking antibodies towards the integrin α3 subunit or integrin α6 subunit, or control IgG (mouse IgG or rat IgG) at 5 µg/ml when plated on growth factor-reduced Matrigel in M199 medium +10% FBS for 43 h. Representative images are shown. b , d The mean numbers of tubes or sprouts in the mouse IgG or rat IgG-treated group are represented as 100%. Dot plots show % mean of the numbers of tubes or sprouts (red lines) and the 95% confidence interval (black lines). Statistical analyses: two-sided Wilcoxon-rank-sum tests. Independent experiments were performed at least two times with similar results. n denotes the number of different fields. b The n for tubes: NECs, n = 12 for mouse IgG and n = 10 for anti-integrin α3; and TECs, n = 12 for mouse IgG and n = 11 for anti-integrin α3. The n for sprouts: TECs, n = 6 for mouse IgG and n = 6 for anti-integrin α3. d The n for tubes: NECs, n = 12 for rat IgG and n = 12 for anti-integrin α6; and TECs, n = 12 for rat IgG and n = 12 for anti-integrin α6. The n for sprouts: TECs, n = 6 for rat IgG and n = 6 for anti-integrin α6. Source data are provided as a source data file.

Article Snippet: For immunofluoresence analysis of mouse glioma, the primary antibodies used were rabbit anti-integrin α3 subunit (Millipore-Sigma, AB1920), rabbit anti-integrin α6 subunit (Bioss, BS-2641R), rabbit anti-CD151 (Epitomics, 5901-1, clone EP6875) and rat anti-mouse CD31 (Dianova, DIA-310) and the secondary antibodies were Alexa-Fluor-568-conjugated goat anti-rabbit IgG (A11036) and Alexa-Fluor-488-conjugated goat anti-rat IgG (A21208) from ThermoFisher Scientific.

Techniques: Incubation, Blocking Assay

Journal: Nature Communications

Article Title: Integrin α3β1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis

doi: 10.1038/s41467-022-31981-2

Figure Lengend Snippet: a Schematic diagram of the experimental procedure. Briefly, GBM tumors (ccf4259, ccf4268, ccf4272, and DI247) cut into ~4 mm in diameter were cultured in Matrigel in neurobasal medium supplemented with 1 ng/ml EGF and 1 ng/ml bFGF. On day 2, anti-integrin α3 subunit blocking antibody or control mouse IgG was injected at three places per tumor piece to achieve ~5 µg/ml in tumor volume. The same injection was repeated on day 6. On day 9, tumors were fixed in 10% formalin and paraffin-embedded. Sections were labeled with rabbit anti-CD31 antibody and Alexa-Fluor-488 goat anti-rabbit IgG, followed by DAPI nuclear stain and imaging. b Representative immunofluorescent images from organotypic culture of tumor ccf4272 are shown. c Percent CD31-positive area per field was obtained by image processing using ImageJ. n denotes the number of different fields, for ccf4269, n = 44 for mouse IgG and n = 95 for anti-integrin α3; for ccf4268, n = 39 for mouse IgG and n = 103 for anti-integrin α3; for ccf4272, n = 57 for mouse IgG and n = 82 for anti-integrin α3; for DI-247, n = 59 for mouse IgG and n = 88 for anti-integrin α3. The boxes indicate the first and third quartiles, bands indicate medians, and the whiskers indicate ±1.5 interquartile range. A linear mixed model was used for statistical analysis ( p < 0.001). Source data are provided as a source data file.

Article Snippet: For immunofluoresence analysis of mouse glioma, the primary antibodies used were rabbit anti-integrin α3 subunit (Millipore-Sigma, AB1920), rabbit anti-integrin α6 subunit (Bioss, BS-2641R), rabbit anti-CD151 (Epitomics, 5901-1, clone EP6875) and rat anti-mouse CD31 (Dianova, DIA-310) and the secondary antibodies were Alexa-Fluor-568-conjugated goat anti-rabbit IgG (A11036) and Alexa-Fluor-488-conjugated goat anti-rat IgG (A21208) from ThermoFisher Scientific.

Techniques: Cell Culture, Blocking Assay, Injection, Labeling, Staining, Imaging

Journal: Nature Communications

Article Title: Integrin α3β1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis

doi: 10.1038/s41467-022-31981-2

Figure Lengend Snippet: a NECs (422 and 623) and TECs (isolates ccf3889, DI-167, and DI-337) were incubated with 25 µg/ml TMR-dex for 30 min, followed by imaging. n denotes the number of different fields: NECs, n = 20 for 422 and n = 20 for 623; and TECs, n = 30 for each isolate. Representative images are shown. Macropinocytosis Index was defined as (total TMR-dex area/total cell area) x 100 per field and Macropinosome Number was defined as the average number of macropinosomes in a cell per field. Statistical analysis: two-sided Wilcoxon-rank-sum tests. Correlation between Macropinocytosis Index and Macropinosome Number was analyzed by linear regression model after adjusting for group effect; shaded area in the correlation graph represents the 95% confidence band for the regression line. b TECs (isolate ccf2390) treated with DMSO (vehicle-control; n = 11) or 100 nM EIPA ( n = 11) for 10 min were subsequently incubated with TMR-dex for 30 min, followed by imaging. Statistical analysis: two-sided Wilcoxon-rank-sum test. c Tube formation of TECs (isolates cf2390 and ccf2687) was quantitated by the numbers of tubes formed per field on Matrigel after 6 h in M199 + 10% FBS in the presence of DMSO (vehicle-control; n = 18 for ccf2390, and n = 18 for ccf2687) or 50 nM EIPA ( n = 18 for ccf2390, and n = 18 for ccf2687). Statistical analysis: two-sided Wilcoxon-rank-sum test. d , e TECs (isolate ccf2390) were pretreated for 3 h with blocking antibody toward integrin α3 ( n = 23) or mouse IgG ( n = 23) and TECs (isolate DI-102) were pretreated with blocking antibody toward CD151 ( n = 11) or mouse IgG ( n = 9), and then TECs were incubated for 30 min with TMR-dex. Statistical analysis: two-sided Wilcoxon-rank-sum test. f Shown are immunoblots of integrin α3 and CD151 from TECs (isolate ccf3889) at 48 h after transfection with siRNA for integrin α3 or CD151. These immunoblots were performed at least two times, and cell lysate samples loaded in separate lanes were from independent replicates. At 48 h after transfection with siRNA, TECs (isolate ccf3889) were incubated with TMR-dex for 15 min. n = number of different fields: integrin α3, n = 16 for siintegrin α3-1 and n = 16 for siintegrin α3-2; CD151, n = 17 for siCD151; and control siRNA, n = 16. Representative images are shown. Statistical analysis: Dunn-method for pairwise-comparisons between each siRNA with ConRi. Boxes indicate the first and third quartiles, bands indicate medians, and whiskers indicate ±1.5 interquartile range. Dotted lines denote means in a – f . Source data are provided as a source data file.

Article Snippet: For immunofluoresence analysis of mouse glioma, the primary antibodies used were rabbit anti-integrin α3 subunit (Millipore-Sigma, AB1920), rabbit anti-integrin α6 subunit (Bioss, BS-2641R), rabbit anti-CD151 (Epitomics, 5901-1, clone EP6875) and rat anti-mouse CD31 (Dianova, DIA-310) and the secondary antibodies were Alexa-Fluor-568-conjugated goat anti-rabbit IgG (A11036) and Alexa-Fluor-488-conjugated goat anti-rat IgG (A21208) from ThermoFisher Scientific.

Techniques: Incubation, Imaging, Blocking Assay, Western Blot, Transfection

Journal: Nature Communications

Article Title: Integrin α3β1 promotes vessel formation of glioblastoma-associated endothelial cells through calcium-mediated macropinocytosis and lysosomal exocytosis

doi: 10.1038/s41467-022-31981-2

Figure Lengend Snippet: a LAMP1 was labelled after TECs (isolate ccf2390) were incubated with 25 µg/ml TMR-dex for 3 h at 37 °C, followed by fixation and permeabilization. Representative images shown; yellow arrows indicate TMR-dex and LAMP1 co-localization. Colocalization was 99% based on the Manders’ Coefficient (M1 = 0.999; M2 = 0.058). Independent experiments were performed at least two-times with similar results. b Experimental-procedure schematic for c , d . Adherent TECs (isolate ccf3889) were incubated with 100 µg/ml TMR-dex for 15 min at 37 °C, washed, harvested, labeled with 5 µg/ml Alexa-Fluor-647-conjugated-WGA in suspension, washed and plated on Matrigel for 20 h. Representative 3-D ( c ) and cross-sectional ( d ) images of tubes. e , f Adherent TECs (isolate ccf2390) were incubated with 100 µg/ml TMR-dex for 15 min at 37 °C, washed, harvested, and plated on Matrigel for 3 h with ( n = 12) or without 0.5 mM EGTA ( n = 10) ( e ), or in the presence of mouse IgG ( n = 21) or integrin α3 antibody ( n = 21) ( f ), followed by fixation and labeling with the LAMP1 mAb, H4A3. g TECs (isolate ccf3889) either in DMEM + 5% FBS (control media) or in calcium-free media were treated with ( n = 20) or without 0.5 mM EGTA ( n = 20) for 15 min, and then incubated with 100 µg/ml TMR-dex for 15 min at 37 °C, and fixed. Representative images are shown for panels e – g . h TECs (isolate ccf3889) in DMEM + 5% FBS were incubated on Matrigel with ( n = 36) or without 0.5 mM EGTA ( n = 36) for 18 h. g , h Statistical analysis: two-sided Wilcoxon-rank-sum tests. n = number of different fields. Boxes indicate first and third quartiles, bands indicate medians, and whiskers indicate ±1.5 interquartile range. Dotted lines denote means. i , j Increase of intracellular Ca 2+ by extracellular Ca 2+ influx was measured by fluorescence intensity of Fluo-8 (Ex/Em = 490/525). To deplete intracellular calcium storage, TECs (isolate DI-102) were treated with 1 µM of Thapsigargin (TH) before addition of extracellular CaCl2 to the medium. Data points indicate means of 3-replicates, and bars denote standard errors. j TECs (isolate DI-102) were preincubated with mouse IgG or blocking anti-integrin α3 antibody, followed by addition of TH and CaCl2. Statistical analysis: linear mixed model. k Proposed TEC tube-formation model. TEC tube-formation mechanism requires macropinocytosis and lysosomal exocytosis. Source data are provided as a source data file.

Article Snippet: For immunofluoresence analysis of mouse glioma, the primary antibodies used were rabbit anti-integrin α3 subunit (Millipore-Sigma, AB1920), rabbit anti-integrin α6 subunit (Bioss, BS-2641R), rabbit anti-CD151 (Epitomics, 5901-1, clone EP6875) and rat anti-mouse CD31 (Dianova, DIA-310) and the secondary antibodies were Alexa-Fluor-568-conjugated goat anti-rabbit IgG (A11036) and Alexa-Fluor-488-conjugated goat anti-rat IgG (A21208) from ThermoFisher Scientific.

Techniques: Incubation, Labeling, Fluorescence, Blocking Assay