Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Extracellular matrix sensing by β1 and β3 integrins controls invadosome formation and localization. (A) Most SrcYF-expressing cells formed invadosome rosettes (visualized by F-actin staining, in green) only on the adhesive surface (gelatin-tetramethylrhodamine B isothiocyanate [TRITC], in red, mixed with vitronectin) and not on antiadhesive areas (Pluronic F127, black areas). (B) Invadosome rosettes stained by phalloidin-TRITC show higher affinity for vitronectin-FITC (light gray bands), sensed by members of the β3 integrin family, than for fibronectin (black bands), sensed by members of both the β1 and β3 integrin families. (C) The β1 and β3 integrins show distinct patterns of localization, observed in MEF-SrcYF cells. β3 (in green in merge) highly colocalized with F-actin, whereas β1 staining is limited to the rosette periphery. (D) 9EG7 antibody β1 staining is specific of the activated form of the integrin. Antibody against activated form of β1 directly conjugated to FITC (9EG7-FITC) was added externally and localized around the invadosome visualized by cortactin-mRFP (in red in merge) expressed in live MEF-SrcYF cells. Bars, 5 μm (A and B), 4 μm (C), and 2 μm (D).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: β1A, and not β3, integrin is essential for invadosome formation. (A) β3 is not essential for invadosome formation and self-assembly into rosettes, visualized by F-actin (in red in merge) and phospho-Y397-FAK (in blue in merge) staining, which occurs in both MEF-SrcYF β3 +/+ or −/− cells. (B) In contrast, β1 depletion in MEF-SrcYF β1 LoxP/LoxP expressing the CRE recombinase for 96 h resulted in the disappearance of isolated invadosomes or in rosettes probed by phalloidin staining. (C) Quantification of the percentage of cells forming invadosomes reveals that almost 95% of MEF-SrcYF β1 LoxP/LoxP treated with CRE recombinase did not form this structure 4 d after infection (n = 650 counted cells/condition). (D) Quantification by qPCR shows an average decrease of 95% in the level of β1 mRNA 96 h post-CRE treatment. Bars, 3 μm (A) and 10 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Staining, Expressing, Isolation, Infection

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: Activation of β1 stimulates invadosome autoassembly. (A) β1 depletion in pOBL-SrcYF induced the disappearance of invadosomes, shown by phalloidin staining of F-actin. Bar, 5 μm. (B) β1 depletion does not affect Src activation. Lysates of pOBL-SrcYF β1 +/+ and −/− cells were probed by Western blotting for phospho-SrcY416, a marker of Src activation, total Src, and actin. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main mutation that activates this integrin. (D) Expression of the preactivated mutants of β1 (β1 D759A) in pOBL-SrcYF β1 −/− cells dramatically increases the number of invadosome rosettes per airy unit, whereas β1WT mutant rescue the rosette number up to the control level.

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Western Blot, Marker, Sequencing, Mutagenesis, Expressing

Journal: Molecular Biology of the Cell

Article Title: β1A Integrin Is a Master Regulator of Invadosome Organization and Function

doi: 10.1091/mbc.E10-07-0580

Figure Lengend Snippet: PKC regulates invadosome autoassembly by phosphorylating Ser785 of β1A integrins. (A) PKC activation by a 60-min treatment of pOBL-SrcYF cells with 2 μM PMA induces a massive increase in invadosome rosette autoassembly, visualized by phalloidin staining. (B) Extracted images from time series (in minutes) from representative observations of pOBL-SrcYF cells expressing GFP-actin and treated with either dimethyl sulfoxide (DMSO; control), the PKC activator PMA (2 μM), and the PKC inhibitor BIM (5 mM). PKC activity regulates the dynamics and maintenance of the invadosome autoassembly state. (C) Amino-acyl sequence of the cytoplasmic domain of β1A integrin and the location of the main PKC targets. (D) Quantification of invadosomes per Airy unit (AU) shows that a mutation mimicking a constitutive phosphorylated form of Ser785 (Asp or Glu) dramatically increases the formation of rosettes in pOBL-SrcYF β1 −/− cells. The nonphosphorylatable mutant of β1 at this site (S785A) has no effect on rosette formation. (E) The number of rosettes/AU was quantified in pOBL-SrcYF cells treated with 2 μM PMA or simultaneously with PMA and 5 mM BIM for 60 min. The inhibitory effect of BIM was determined by calculating the percent inhibition of invadosome rosette formation. Mutants mimicking a constitutively phosphorylated Ser785 (S785D and S785E) show only 50% inhibition after BIM treatment, indicating that this residue on β1 is a major target of PKC in the regulation of invadosome autoassembly. Bars, 20 μm (A) and 5 μm (B).

Article Snippet: Antibodies for immunoblotting and immunofluorescence were obtained from the following commercial sources: rabbit anti-phospho-Tyr 418 Src and anti-phospho-Tyr397 FAK (Invitrogen, Carlsbad, CA), mouse anti-Src (GD11; Millipore, Billerica, MA), mouse anti-actin (Sigma-Aldrich, St. Louis, MO), mouse anti-paxillin (BD Biosciences, Franklin Lakes, NJ), mouse anti-human β1 integrin (clone 4B7R; Becton Dickinson, le Pont de Claix, France), rat anti-mouse β1 integrin (MB1.2; Millipore), and rat anti-mouse β3 integrin (clone LucA5; Emfret Analytics, Würzburg, Germany).

Techniques: Activation Assay, Staining, Expressing, Activity Assay, Sequencing, Mutagenesis, Inhibition

Journal: Arthritis and rheumatism

Article Title: Role of Semaphorin 7a signaling in TGF-?1 induced lung fibrosis and scleroderma-related interstitial lung disease

doi: 10.1002/art.30386

Figure Lengend Snippet: Sema-7a expression by bone marrow derived cells is sufficient for fibrosis and fibrocyte accumulation in the TGF-β1 exposed lung. A. BAL cell counts in TGF-β1 Tg+ mice with the Sema-7a locus intact (left comparisons) or deleted (right comparisons). B. Total left lung collagen content, same comparisons. Transplantation with Sema-7a null donor marrow did not significantly reduce collagen accumulation while transplantation with WT bone marrow was sufficient to restore collagen accumulation in the TGF-β1 x Sema-7a null mice. C. CD45+Col-Iα+ cells, same comparisons. As with the collagen content, transplantation of TGF-β1 x WT mice with Sema-7a null mice did not reduce fibrocytes. However, transplantation of TGF-β1 x Sema-7a null mice with WT marrow restored fibrocyte accumulation to wild type levels. D. Expression of Plexin C1 (left) and β1 integrin (right) in TGF-β1 x Sema-7a recipients of marrow with the Sema-7a locus disrupted or intact. Replacement of Sema-7a on BM-derived cells is accompanied by a trend towards increased expression of β1 integrin. *p<0.05. **p<0.01.

Article Snippet: Primers specific for human Sema-7a, β1 integrin subunit, Plexin C1 and GAPDH, and murine β1 integrin subunit, Plexin C1, and β-actin were purchased from Superarray Bioscience.

Techniques: Expressing, Derivative Assay, Transplantation Assay

Journal: Arthritis and rheumatism

Article Title: Role of Semaphorin 7a signaling in TGF-?1 induced lung fibrosis and scleroderma-related interstitial lung disease

doi: 10.1002/art.30386

Figure Lengend Snippet: Expression of Sema-7a (A) is increased in the blood of patients with SSc-ILD compared to normal controls and patients with SSc that lack ILD. β1 integrin is increased in SSc-ILD patients compared to control (B). Expression of Plexin C1 (C) is not significantly increased. White bar: normal subjects. Grey striped bar: SSc without ILD. Black bar: SSc-ILD. *p<0.05. **p<0.01.

Article Snippet: Primers specific for human Sema-7a, β1 integrin subunit, Plexin C1 and GAPDH, and murine β1 integrin subunit, Plexin C1, and β-actin were purchased from Superarray Bioscience.

Techniques: Expressing

Journal: Cancer Science

Article Title: Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer

doi: 10.1111/cas.14566

Figure Lengend Snippet: Identification of integrin α3 as the target antigen of OV‐Ab 30‐7. A, Silver staining of proteins eluted from OV‐Ab 30‐7‐conjugated protein G affinity column. E1, E2: elution supernatants from protein G‐OV‐Ab 30‐7 affinity column. Ab only: 5 μg/mL OV‐Ab 30‐7. B, Peptide sequences of the target protein were deduced from MS/MS analysis. Identified peptides are shown in red. C, SKOV‐3 cell lysates were immunoprecipitated with normal mouse (NM) IgG, MAB2056 commercial anti‐integrin α3 antibody, or OV‐Ab 30‐7 antibody. Western blotting was performed with rabbit anti‐integrin α3 antibody. D, Flow cytometric analysis of OV‐Ab 30‐7 binding to ITGA3 and luciferase knockdown SKOV‐3 cells. Cells (2 × 10 5 per condition) were incubated with different concentrations of OV‐Ab 30‐7 and detected with PE‐conjugated anti‐mouse IgG. * P < .05

Article Snippet: SKOV‐3 cell lysates and recombinant integrin α3 (#TP320975, OriGene, Rockville, MD, USA) or integrin β1 (#H00003688‐P01, Abnova, Taipei, Taiwan) were applied to OV‐Ab 30‐7 or NM IgG‐coupled Protein G Sepharose (GE Healthcare Biosciences, Boston) for l h. Then the beads were washed 4 times with PBS containing 1% Tween‐20.

Techniques: Silver Staining, Affinity Column, Tandem Mass Spectroscopy, Immunoprecipitation, Western Blot, Binding Assay, Luciferase, Knockdown, Incubation

Journal: Cancer Science

Article Title: Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer

doi: 10.1111/cas.14566

Figure Lengend Snippet: OV‐Ab 30‐7 induces apoptosis, p53 upregulation, caspase activation, and PARP cleavage in SKOV‐3 cells. A, Apoptosis of SKOV3‐shLuc, ES‐2, NNM, and SKOV‐3 integrin α3‐knockdown cells. FITC‐annexin‐V and PI staining were used to detect apoptosis after incubation with 0.2, 2, or 20 μg of OV‐Ab 30‐7 or NM IgG for 6 h. * P < .05, ** P < .01. B, Flow cytometry analysis of caspase‐3, 8, and 9 activation in SKOV‐3 cells. Cells were treated with NM IgG or OV‐Ab 30‐7 for 6 h at the indicated concentrations. Representative histograms are shown, with quantitative results below. C, Western blot analysis of cleaved PARP in SKOV‐3 cells. Cells were treated with OV‐Ab 30‐7 or NM IgG (40 μg/mL) for 24, 48, and 72 h. D, Western blot analysis of p53 and p21. Cells were treated with OV‐Ab 30‐7 or NM IgG for 30 min. E, Representative images of ES‐2 (upper) and SKOV‐3 (lower) cells treated with 20 μg/mL OV‐Ab 30‐7 or NM IgG. Quantification of the spheroid diameters is shown to the right. Scale bar, 200 µm

Article Snippet: SKOV‐3 cell lysates and recombinant integrin α3 (#TP320975, OriGene, Rockville, MD, USA) or integrin β1 (#H00003688‐P01, Abnova, Taipei, Taiwan) were applied to OV‐Ab 30‐7 or NM IgG‐coupled Protein G Sepharose (GE Healthcare Biosciences, Boston) for l h. Then the beads were washed 4 times with PBS containing 1% Tween‐20.

Techniques: Activation Assay, Knockdown, Staining, Incubation, Flow Cytometry, Western Blot

Journal: Cancer Science

Article Title: Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer

doi: 10.1111/cas.14566

Figure Lengend Snippet: Integrin α3 is co‐expressed with laminin and OV‐Ab 30‐7 can block laminin‐integrin signaling. A, IHC staining of integrin α3 and laminin‐α5 in ovarian cancer tissue reveals co‐expression of the proteins. Scale bars, 100 μm (×200). B, Cell adhesion assay. SKOV‐3 and ES‐2 cells were seeded on laminin, fibronectin or collagen‐coated plates for 45 min. After washing, adherent cells were quantified relative to control. C, Transwell migration assay. SKOV‐3 cells were treated with PBS, NM IgG, or OV‐Ab 30‐7 (20 μg/mL), then seeded on laminin‐coated wells for 8 h. D, SKOV‐3 and ES‐2 cells were pre‐incubated with OV‐Ab 30‐7, NM IgG for 15 min at 37°C, then seeded on plates with or without laminin coating for 30 min. Western blot analysis of the indicated proteins was performed. Right panel shows the quantification of phosphorylated FAK signal from western blots

Article Snippet: SKOV‐3 cell lysates and recombinant integrin α3 (#TP320975, OriGene, Rockville, MD, USA) or integrin β1 (#H00003688‐P01, Abnova, Taipei, Taiwan) were applied to OV‐Ab 30‐7 or NM IgG‐coupled Protein G Sepharose (GE Healthcare Biosciences, Boston) for l h. Then the beads were washed 4 times with PBS containing 1% Tween‐20.

Techniques: Blocking Assay, Immunohistochemistry, Expressing, Cell Adhesion Assay, Control, Transwell Migration Assay, Incubation, Western Blot

Journal: Cancer Science

Article Title: Novel monoclonal antibody against integrin α3 shows therapeutic potential for ovarian cancer

doi: 10.1111/cas.14566

Figure Lengend Snippet: Integrin α3 and laminin expression in ovarian cancer correlate with poor prognosis. A, IHC staining of integrin α3 in ovarian cancer and tumor‐adjacent normal ovary tissue. B, Positive and negative integrin α3 IHC staining in the different types of ovarian cancer. C, Overall survival of ovarian cancer patients from the TCGA database stratified by expression of ITGA3, ITGB1, or both. Gene expression was determined by RNA sequencing and reported as median number Fragment Per Kilobase of exon per Million (FPKM) reads. The cut‐off value for ITGA3 was 35.34, and for ITGB1, it was 44.09 FPKM. D, In total, 31 clinical ovarian cancer specimens were analyzed by IHC for laminin‐α5 and integrin α3. Representative images of weak, moderate, and strong staining are shown. The number of cases with each staining intensity is indicated in the table below ( P < .01). E, Relationships between ITGA3 and laminin‐related gene expression in patients from the TCGA database. Data were visualized by cBioPortal ( https://www.cbioportal.org/ ) and evaluated with Pearson’s correlation analysis. F, Kaplan‐Meier survival curves of ovarian cancer patients stratified by LAMA3, LAMB3, and LAMC2 expression. G, Kaplan‐Meier survival curves for ovarian cancer patients stratified by ITGA3 expression combined with laminin‐related gene (LAMA3, LAMB3, LAMC2) expression

Article Snippet: SKOV‐3 cell lysates and recombinant integrin α3 (#TP320975, OriGene, Rockville, MD, USA) or integrin β1 (#H00003688‐P01, Abnova, Taipei, Taiwan) were applied to OV‐Ab 30‐7 or NM IgG‐coupled Protein G Sepharose (GE Healthcare Biosciences, Boston) for l h. Then the beads were washed 4 times with PBS containing 1% Tween‐20.

Techniques: Expressing, Immunohistochemistry, Gene Expression, RNA Sequencing, Staining

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is necessary for epiblast cell survival upon implantation and pluripotency transition (A–C) Analysis of apoptosis by cleaved caspase-3 staining in wild-type (fl/fl, top) and mutant (Δ/Δ, bottom) mESC spheroids at 24 (A), 48 (B), and 72 h (C) of culture. Steady increase in the number of c-caspase-3 + structures in mutants over time. Fisher’s exact test: (A) p = ns (number of spheroids n = 42 [fl/fl], n = 44 [Δ/Δ]); (B) ∗∗∗∗ p < 0.0001 (n = 38 [fl/fl], n = 48 [Δ/Δ]); and (C) ∗∗∗∗ p < 0.0001 (n = 26 [fl/fl], n = 28 [Δ/Δ]). (D–G) In vivo -recovered post-implantation embryos at E5.5, as shown in Figure S1 D: epiblast is either fl/fl or Δ/Δ; visceral endoderm (VE) and extraembryonic ectoderm (ExE) are +/+. In contrast to wild-type embryos (Epi:fl/fl) (D), embryos deficient for integrin β1 (Epi:Δ/Δ) either have a small epiblast (E), show few residual Oct4 + cells (F), or lack epiblast completely. Quantification of the number of embryos with epiblast versus degenerated/lethal—Fisher’s exact test: ∗∗∗ p = 0.0003 (number of embryos n = 15 [fl/fl], n = 25 [Δ/Δ], 4 replicates). (H–J) Assessment of apoptotic cells in chimeric blastocysts at E4.5–4.75, Epi:fl/fl (H, top) or Epi:Δ/Δ (H, bottom): embryos are sequentially re-stained to assess NANOG cells in the c-caspase-3 channel. Mutant embryos (Epi:Δ/Δ) contain a significantly higher number of apoptotic cells within the epiblast compartment: quantification of the percentage of c-caspase-3 + cells over total epiblast cell number (I), and percentage of apoptotic cells in wild type (median = 1.7%) versus mutants (median = 14%), Mann-Whitney test; ∗∗∗∗ p < 0.0001 (number of embryos n = 37 [fl/fl], n = 29 [Δ/Δ]). Correlation between the percentage of c-caspase-3 + cells with the percentage of either OTX2 (top) or NANOG + cells (bottom) within the epiblast (J): exit from naive pluripotency (increase in OTX2 and decrease in NANOG cells) correlates with the increase in the percentage of c-caspase-3 + cells (number of embryos n = 23 [fl/fl], n = 15 [Δ/Δ]). (K and L) Wild-type and mutant mESCs display comparable minimal levels of apoptotic cells when cultured in 2iLIF (naive pluripotency status, absence of OTX2 expression). (K) Fisher’s exact test: p = ns (number of spheroids n = 33 [fl/fl], n = 37 [Δ/Δ]). In the absence of 2iLIF (L), cells transit to formative pluripotency as shown by the expression of OTX2 and activate the apoptotic pathway in the absence of integrin β1 (Δ/Δ). Scale bars: 5 μm (A), 10 μm (B, C, K, and L), 15 μm (H, inset), 25 μm (H, inset, and D–F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Staining, Mutagenesis, In Vivo, MANN-WHITNEY, Cell Culture, Expressing

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Fine-tune spatial segregation between integrin β1 and actomyosin (A) Configuration of the mouse epiblast before implantation (E4.5): cells are apolar and display heterogenous localization of integrin β1 (Itg-β1) and actomyosin, as shown by the distribution of phalloidin (F-actin) and phosphorylated non-muscle myosin (pMLC-II). (B) Configuration of the mouse epiblast upon implantation (E5.0): segregation in a mutually exclusive manner between integrin β1 and actomyosin while cells acquire a wedge-shape morphology. (C) Configuration of the mouse epiblast at post-implantation (E5.5): cells of the mature epiblast epithelium are columnar in shape surrounding a central pro-amniotic cavity. Integrin β1 localizes on the basolateral domain and actomyosin on the apical side. (D) Integrin β1 and actomyosin are heterogeneously distributed in mouse embryonic stem cells (mESCs) maintained in naive pluripotency (+2iLIF, left panel), similarly to E4.5. Integrin β1 and actomyosin become spatially segregated during formative pluripotency following 72 h of removal of 2iLIF (−2iLIF, right panel), similarly to E5.5. Scale bars: 25 μm (A–D). Magnified/inset areas indicated by arrows (A and B).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques:

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Loss of integrin β1 causes basal actomyosin accumulation and morphogenesis failure (A and B) Distribution of actomyosin in mESC after 24 (A) and 48 h of culture (B). Wild-type cells (fl/fl) display enrichment of F-actin and p-myosin at the central point of the apical membrane initiation site (AMIS) at 24 h (A, top). Apical actomyosin is maintained after 48 h at the center of the rosette configuration (B, top). Mutant cells (Δ/Δ) display ectopic accumulation of actomyosin basally (A, bottom), giving rise to a large basal actomyosin cable after 48 h of culture (B, bottom). (C) Blebs and ectopic E-cadherin junctions appear on the basal domain of mutant mESCs (arrows). (D) Distribution of actomyosin in the mouse blastocyst at E4.5, chimeric for either fl/fl (top) or Δ/Δ (bottom) alleles of Itgβ1 . Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the central point of apical constriction, forming a rosette configuration (top). Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the rosette (bottom). (E) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E4.5 shows significant increase of basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 21 [fl/fl], n = 23 [Δ/Δ]). (F) Assessment of actomyosin localization in post-implantation embryos at E5.5. Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the apical side of the epiblast epithelium. Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the central pro-amniotic cavity. (G) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E5.5: significant increase in basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). (H) Quantification of percentage of embryos forming cavity in the epiblast at E5.5: all mutant epiblasts fail to form the cavity: Fisher’s exact test: ∗∗∗∗ p = 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). Scale bars: 5 μm (A), 10 μm (B and C), 25 μm (D), and 50 μm (F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Membrane, Mutagenesis, Fluorescence, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is dispensable for the establishment but necessary for the maintenance of apicobasal polarity (A) Distribution of the polarity marker PAR3 in mESCs at 1- and 2-cell stage (24 h of culture in −2iLIF). Wild-type cells (fl/fl, top) recruit PAR3 toward the AMIS similarly to mutant cells (Δ/Δ, bottom). (B) Quantification of the orientation of apicobasal polarity at 24 h: Fisher’s exact test: p = ns (number of mESC spheroids n = 42 [fl/fl], n = 39 [Δ/Δ]). (C and D) Distribution of centrosomes, as shown by γ-tubulin staining, at 2-cell stage and quantification of the angles along the nuclear-centrosome axis. Centrosome-nuclear axis angle: means ± SEMs (red). Test: Mann-Whitney test: p = ns (number of mESC spheroids n = 26 [fl/fl], n = 31 [Δ/Δ]). (E and F) Assessment of polarization in agarose at 24 h by Golgi and PAR3 localization. Fisher’s exact test: p = ns (number of spheroids n = 38 [fl/fl], 38 [Δ/Δ]). Both wild-type and mutant cells display correct apicobasal polarity at 24 h of culture, even in the absence of ECM components. (G) Assessment of polarization at 48 h. PAR6 is recruited at the apical site where actomyosin accumulated in wild type. PAR6 is recruited basally at the site of actomyosin accumulation in mutant cells. (H) In wild type, podocalyxin vesicles are recruited apically at PAR6 site. In mutant, PAR6 localizes basally, and podocalyxin vesicles are secreted basally at this latter site. (I) Quantification of the orientation of apicobasal polarity at 48 h, assessed by Golgi and PAR6 localization. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (n = 30 [fl/fl], n = 29 [Δ/Δ]). Scale bars: 5 μm (A, C, and E) and 10 μm (G and H).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Marker, Mutagenesis, Staining, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vitro rescue of integrin-mediated survival and morphogenesis via FGF/IGF stimulation and ROCK inhibition (A) ROCK inhibition by Y27632 rescues lumen initiation in mESCs deficient for integrin β1 (Δ/Δ, bottom): Golgi is correctly oriented apically, podocalyxin vesicles are secreted apically, polarity marker aPKC is recruited at the apical domain, and actomyosin is re-established apically. Quantification of the percentage of mESC spheroids undergoing lumenogenesis shows no significant differences between wild-type and mutant cells: Fisher’s exact test: p = ns (number of spheroids n = 65 [fl/fl], n = 68 [Δ/Δ]). (B) Assessment of structures showing apoptotic cell death following ROCK inhibition treatment: mutant cells activate apoptosis despite rescue of polarity and lumen formation. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 35 [fl/fl], n = 39 [Δ/Δ]). (C) Stimulation of the survival pathways via supplementation of FGF2, IGF1, and GSK3i is not sufficient to prevent initiation of apoptosis in integrin β1 mutant cells (Δ/Δ): Fisher’s exact test: ∗∗∗ p = 0.0002 (n = number of spheroids = 35 [fl/fl], n = 35 [Δ/Δ]). (D) Inhibition of ROCK coupled to supplementation of FGF2, IGF1, and GSK3i restores both morphogenesis and survival in spheroids deficient for integrin β1 (Δ/Δ, bottom): Fisher’s exact test: p = ns (number of spheroids n = 47 [fl/fl], n = 40 [Δ/Δ]). (E and F) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids cultured for 48 h in medium only shows myosin ectopic localization on the basal side of mutant cells (ratio <1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 31 [fl/fl], n = 30 [Δ/Δ]). (G and H) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids supplemented with FGF2, IGF1, GSK3i, and ROCKi for 48 h shows re-establishment of myosin in the apical domain in mutant cells (ratio >1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗ p = 0.002 (number of spheroids n = 35 [fl/fl], n = 31 [Δ/Δ]). Despite the increase in the ratio of apicobasal p-myosin, mutants still differ significantly from wild types. Scale bars: 10 μm (A–D, F, and H),

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vitro, Inhibition, Marker, Mutagenesis, Fluorescence, Cell Culture, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vivo rescue of survival and morphogenesis in mouse embryos and spatial segregation of integrin β1 and actomyosin in the human embryo (A) Culture of mouse embryos from pre- to post-implantation as shown in Figure S3 F: wild-type controls (Epi:fl/fl) develop into post-implantation egg-cylinders (left), mutant epiblasts deficient for integrin β1 (Epi:Δ/Δ) fail to undergo lumenogenesis and to survive during post-implantation development in normal culture conditions (center). Supplementation with ROCKi, FGF2, IGF1, and GSK3i restores lumenogenesis and survival of the epiblast compartment in integrin β1-deficient embryos (right). (B) Quantification of the percentage of embryos undergoing lumenogenesis in wild-type and mutant embryos cultured in normal conditions compared to mutant embryos cultured in the presence of ROCKi, FGF2, IGF1, and GSK3i. Fisher’s exact test: fl/fl versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i p = ns; Δ/Δ medium versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i ∗∗∗∗ p < 0.0001 (number of embryos n = 26 [fl/fl], n = 16 [Δ/Δ], n = 51 [Δ/Δ, ROCKi/FGF2/IGF1/GSK3i]). (C) Schematic summary of results. Wild-type (left): integrin β1-mediated adhesion to the basement membrane leads to suppression of actomyosin basally, allowing its apical localization. Activation of actomyosin at the apical side leads to the apical localization of PAR6 ( Figure 4 G) and secretion of podocalyxin vesicles and initiation of lumenogenesis ( Figure 4 H). Integrin β1 promotes epiblast survival via stimulation of FGF/ERK and IGF1/AKT pathways. Mutant (right): loss of integrin β1 leads to ectopic actomyosin accumulation basally. The basal actomyosin recruits PAR6 basally, which directs podocalyxin vesicles toward the basal side, preventing lumenogenesis. Loss of integrin β1 leads to apoptosis. (D) Assessment of the localization of integrin β1 and actomyosin in human embryos at day 8 d.p.f. shows initiation of spatial segregation between the 2 complexes in a mutually exclusive manner (arrows). At 10 d.p.f., the 2 are fully segregated, with integrin β1 localizing on the basolateral domain, while actomyosin is confined on the apical side of the epiblast epithelium facing the central amniotic cavity. Scale bars: 25 μm (A) and 50 μm (D).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vivo, Mutagenesis, Cell Culture, Membrane, Activation Assay

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet:

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Recombinant, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG protein is associated with β1 integrin and activates β1 integrin. a - b . Coimmunoprecipitation. Cell membrane proteins of HCT116 cells were collected and subjected to immunoprecipitated (IP) using anti-SNCG ( a ), anti-SNCG or anti-β1 integrin antibody ( b ). The IP proteins or total cell lysates were analyzed by Western blot. Normal IgG served as the negative control. c . Far-Western blot analysis. HCT116 cells were transfected with control siRNA (lane 1-2), and specific siRNA-β1-2 (lanes 3-4) for 48 h. Cells were treated without (lane 1, 3) or with 1 μmol/L rhSNCG (lane 2, 4). Cell lysates were subjected to SDS-PAGE and transferred to NC membrane. β1 integrin (prey protein) on the membrane is detected with SNCG (bait protein). More SNCG was associated with membrane β1 integrin in SNCG-treated cells than that in the control cells (lane 1, 2). Correspondingly, less SNCG were detected in β1 integrin knock-down cells than that in control cells (lane 2, 4). d - e , Effect of concentration and time treatment of SNCG on activated β1 integrin. HCT116 cells were stimulated with GST or GST-SNCG at various concentrations for 60 min ( d ) or at fixed concentration (1 μmol/L) for various times ( e ). Cell lysates were analyzed with the HUTS-21 mAb recognizing the activated form of β1 integrin. f , GST-SNCG treatment (1 μmol/L) upregulated activated β1 integrin subunit in HCT116 and SW480 cells. g , Colocalization of SNCG with F-actin. HCT116 cells grown on coverslips were transiently transfected with control siRNA or β1-specific siRNA-2. After 72 h, cells were treated with GST or GST-SNCG (1 μmol/L) for 60 min. Cells were fixed and stained with anti-SNCG (red) and FITC-Phalloidin (green). Colocalization of SNCG and F-actin was shown in yellow. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Immunoprecipitation, Western Blot, Negative Control, Far Western Blot, Transfection, SDS Page, Concentration Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Integrin β1 is required for enhancement of SNCG on tumor cell migration and invasion. a - b , The functional blocking antibody for β1 integrin subunit (5, 10, 20 μg/mL) was added in the upper compartment of migration or invasion chambers stimulated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( a ) or 48 h for invasion ( b ). c - d , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. then cells were treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( c ) or 48 h for invasion ( d ). e - f , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min and then treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( e ) or 48 h for invasion ( f ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. g , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot. h , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Functional Assay, Blocking Assay, Transfection, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: FAK is essential for SNCG-enhanced tumor cell migration and invasion. a - b , HCT116 cells were transfected with control siRNA and FAK-specific siRNA-2, -3 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for migration ( a ) or invasion ( b ). c - d , HCT116 cells were treated with PBS, 50 μmol/L FAK inhibitor 14 for 30 min and then treated with or without GST-SNCG (1 μmol/L) for migration ( c ) or invasion ( d ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. e - f , HCT116 cells were transfected with control siRNA (lane 1-2), and FAK-specific siRNA-2 (lane 3-4) and -3 (lane 5-6) for 72 h. cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed for activated and total β1 integrin ( e ) or activated and total FAK ( f )

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Migration, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG is an indicator of adverse prognosis and positively correlates with activated β1 integrin, p-FAK (Y 397 ) in CRC tissues. a - b , Kaplan-Meier estimation of disease-free survival (DFS) for stage I-II ( a ) and III-IV ( b ) colorectal adenocarcinoma patients according to SNCG levels. c , Correlations of SNCG levels in CRC tissues with post-operative recurrence and status. d , Representative blots from three independent experiments were presented. Protein levels of SNCG, activated β1 integrin, and p-FAK (Y 397 ) in clinical colon cancer tissue samples were evaluated by Western blot analysis. In order to increase the reproducibility, HCT116 cell lysates were used in each blot as the internal control ( c ) to minimize the effect of band intensity variation. GAPDH was used as the loading control. e - g , Correlation between the relative protein levels of activated β1 integrin levels and p-FAK (Y 397 ) ( e ), SNCG and active β1 integrin ( f ), and SNCG and p-FAK (Y 397 ) ( g ) were plotted as a scatter plots

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Exogenously added SNCG remodels the microenvironment of tumor cells and increases MMP-2 activity by β1 integrin. a , Antibody array screening of CM from GST and GST-SNCG-treated HCT116 cells. b , CM (left panel) and whole cell lysates (right panel) from HCT116 and SW480 cells treated with or without GST-SNCG (1 μmol/L) were subjected to Western blot analysis. Representative blots from three independent experiments were presented. c - d , HCT116 cells were treated with diluent, 50 μmol/L MMP-2 inhibitor for 40 min, then 1 μmol/L GST or GST-SNCG was added in the cell medium for migration ( c ) or invasion ( d ) assay. Migrated or invaded cells were quantitated after 24 h or 48 h, respectively. Error bars, SE of three determinations. e - f , Western blot and gelatin zymography analysis. CM from HCT116 cells treated with GST or GST-SNCG (1 μmol/L) in β1 integrin knock-down ( e ) or RGD-treated cells ( f ) was analyzed by gelatin zymography with FBS as the positive control, and secreted protein levels were analyzed by Western blot

Article Snippet: Mouse anti-human β1 integrin monoclonal antibody (mAb) (specifically recognizing the active conformation) is from BD Biosciences.

Techniques: Activity Assay, Ab Array, Western Blot, Migration, Zymography, Positive Control

Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet: ( a ) Upper panels, total flow cytometry plots of HUVEC and TIME cells stained for endothelial cell markers PECAM and von Willebrand factor (vWF). Medium and lower panels, surface flow cytometry plots of HUVEC and TIME cells stained for PCDH1, β3 integrin, DAF, β1 integrin. ( b ) Surface flow cytometry of wild-type (WT) and knockout (KO) TIME cells stained as above. Histograms of WT cells are shown in gray; single- and double-KO cells are shown in color. ( c ) Western blot analysis of WT TIME cells and KO cells ± cDNA. β-Actin was used as a loading control. Figure 1—source data 1. Original blot of WT TIME cells and KO cells ± cDNA.

Article Snippet: Antibody , APC-α-Human-β1-Integrin (Mouse monoclonal) , BD , Cat. # 59883 , Flow 1:20.

Techniques: Flow Cytometry, Staining, Knock-Out, Western Blot

Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet:

Article Snippet: Antibody , APC-α-Human-β1-Integrin (Mouse monoclonal) , BD , Cat. # 59883 , Flow 1:20.

Techniques: Plasmid Preparation, Expressing, Transduction, Recombinant, Sequencing, Staining, Software, Imaging