Journal: International Journal of Biological Sciences

Article Title: Sema3d hi Fibroblasts Promote Acute Kidney Injury Fibrotic Progression Through Confining Endothelial Cell Migration

doi: 10.7150/ijbs.124971

Figure Lengend Snippet: Effects of Sema3d hi -NRK49F on integrin internalization and Arf6 activation in endothelial cells. (A) Western blot detection of integrin β1 in HUVEC stimulated with conditioned medium from NRK-49F, NRK49F-Vector and NRK49F-OE cells and quantification results. n = 3. (B) Immunofluorescence staining of integrin β1 in HUVEC stimulated by the conditioned medium from NRK-49F, NRK49F-Vector and NRK49F-OE cells. Scale bar = 20 μm. (C) Immunofluorescence staining of integrin β1 in HUVEC stimulated by control medium and 10nM Sema3d recombinant protein. Scale bar = 20 μm. (D) Effects of conditioned medium fromNRK49F-Vector and NRK49F-OE cells on the activation of Arf6 in HUVEC. The left shows the pull-down experiment flowchart, the middle shows the western blot results, and the right provides statistical results, n = 3. ns: no significance, ***p < 0.001.

Article Snippet: Anti-β1 integrin antibody (sc-18887, Santa Cruz, USA) was diluted to a final concentration of 5 μg/mL in DMEM containing 0.01% BSA and pre-cooled at 4°C.

Techniques: Activation Assay, Western Blot, Plasmid Preparation, Immunofluorescence, Staining, Control, Recombinant

Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Increased integrin α5 expression correlates with enhanced bacterial adherence and uptake in vinculin -/- cells. (A) Vinculin WT and vinculin -/- cells were seeded on fibronectin coated glass bottom imaging dishes, next day fixed and stained for vinculin expression using α-human vinculin antibody (clone hVIN1). Total amounts of integrins were stained using integrin β1 antibody (clone HMβ1-1). TIRF microscopy was used to assess the localization of vinculin and β1 integrins. Bars represent 10 μm. (B) Equal amounts of protein in the whole cell lysates (WCL) of vinculin WT and vinculin -/- cells were separated by SDS-PAGE and processed for Western blotting using antibodies against talin, FAK, vinculin, paxillin, Src, or β-tubulin as indicated. Representative blots are shown. (C and D) Cells were seeded on gelatine coated 24 well plates and infected with S. aureus or S. carnosus with MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D) , respectively. Values are means ± SEM from 5 independent experiments done in quadruplicate (n=20). Statistical significance of data was assessed by Mann-Whitney test. (E) Cells were stained for surface expression of β1, α5, αv, or β3 integrins and examined by flow cytometry. Data are shown as mean fluorescence intensity (MFI) and are corrected for the background MFI of integrin β1 -/- fibroblasts (β1 integrin staining) or corrected for the MFI of cells stained in the absence of primary antibody (α5, αv, and β3 integrin stainings). Results are shown as mean ± SEM from 3 independent experiments. (F) mRNA levels of β1, α5, αv, or β3 integrin transcripts in vinculin WT or vinculin -/- cells were analyzed by qRT-PCR. Integrin expression was normalized to GAPDH expression and was set to 1 for vinculin WT cells. Bars represent the mean of two independent experiments each performed in triplicate.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Expressing, Imaging, Staining, Microscopy, SDS Page, Western Blot, Infection, Bacteria, MANN-WHITNEY, Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Transient vinculin overexpression in 293 cells does not interfere with bacterial uptake. (A) 293 cells were transfected with constructs encoding GFP or GFP-vinculin. 48 h post-transfection whole cell lysates (WCLs) were prepared and analyzed for vinculin and β-tubulin expression by immunoblotting. Representative blots are shown. (B) Cells transfected as in (A) were compared for integrin α5 surface expression by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibody and a rhodamine-coupled secondary antibody (blue – GFP; red – GFP-vinculin) or cells stained with the secondary antibody only (grey lines). (C and D) Cells as in (A) were infected with S. aureus and S. carnosus for 2 h (MOI 20). Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D) , respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical significance testing.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Over Expression, Transfection, Construct, Expressing, Western Blot, Flow Cytometry, Staining, Infection, Bacteria, MANN-WHITNEY

Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Knock-down of vinculin in wildtype fibroblasts does not affect S. aureus uptake. (A) Murine fibroblasts were transduced with lentiviral particles harboring either pLKO.1 or pLKO.1 encoding shRNA against mouse vinculin (pLKO-1 sh-mvinc). WCL of the stably transduced cell lines were analyzed for FAK, vinculin, paxillin, Src and β-tubulin protein expression by Western blotting. Representative blots are shown. (B) Integrin α5 surface expression in cells from (A) was analyzed by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibodies and a rhodamine-coupled secondary antibody (blue – pLKO.1; red – pLKO.1 sh-mvinc) or cells stained with the secondary antibody only (contr.; grey lines). (C) pLKO.1 or pLKO.1 sh-mvinc cells were seeded on gelatine coated 24 well plates. Next day, cells were infected with S. aureus at MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (top) or gentamicin protection assays (bottom), respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical testing.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Knockdown, Transduction, shRNA, Stable Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Infection, Bacteria, MANN-WHITNEY

Journal: Cell Host & Microbe

Article Title: β1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections

doi: 10.1016/j.chom.2017.05.001

Figure Lengend Snippet:

Article Snippet: The samples were stained with a rat anti-mouse β1-integrin (1:100 dilution, clone MB1.2, Merck Millipore), anti-acid ceramidase (1:100 dilution), anti-ceramide (1:100 dilution), anti-sphingosine (1:1000 dilution), anti-mouse β2-integrin antibodies (1:100, clone M1812, 1:100, #557437, BD) or FITC-Annexin (1:200, #11 828 681 001, Roche) in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl 2 , 0.7 mM MgCl 2 , 0.8 mM MgSO 4 ) plus 1% FCS at room temperature for 45 min.

Techniques: Virus, Isolation, Recombinant, Software

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Domain architecture of an active (open headpiece/extended) integrin α5β1 heterodimer. α5 Subunit is grey; β1 subunit headpiece and leg are, respectively, in shades of blue and green. The localization of three different mAb epitopes, exposed only in the conformationally active β1 subunit, is represented. Epitopes of mAb 12G10 and mAb HUTS4, respectively, lie in the βI domain and hybrid domain of the headpiece, whereas mAb 9EG7 epitope is in the I-EGF2 domain. (B) Confocal immunofluorescence microscopy analysis of the subcellular localization of the three different anti-active β1 integrin mAbs employed to stain fixed ECs. All three mAbs bind to active β1 integrins mainly located within typical elongated fibrillar adhesions. Scale bar 20 µ m; magnification scale bar 10 µ m. (C) Confocal immunofluorescence microscopy analysis of anti-active β1 integrin mAbs localization after 10 min of incubation on living ECs. Anti-I-EGF2 domain mAb 9EG7 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, whereas anti-βI domain mAb 12G10 recognizes active β1 integrins located both outside and inside highly fragmented and tiny adhesions. Similar to mAb 9EG7, the anti-hybrid domain mAb HUTS4 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, hinting that anti-active headpiece mAb-elicited fragmentation specifically depends on βI domain binding. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Selected frames from (top row) and (bottom rows), respectively, illustrating dynamic mAb 9EG7-Alexa Fluor 488 binding to active β1 integrins over time upon live incubation on ECs either in the absence (top row) or in the presence (bottom rows) of mAb 12G10–Alexa Fluor 647. When incubated alone (top row), mAb 9EG7–Alexa Fluor 488 preferentially binds active β1 integrins located within fibrillar adhesions and remains stable over time. When mAb 12G10–Alexa Fluor 647 is pre-incubated on ECs, mAb 9EG7–Alexa Fluor 488 does no longer localize in fibrillar adhesions. Scale bar 20 µ m. (E) Representative g -STED confocal microscopy pictures of anti-active β1 integrin 9EG7 mAb localization after 10-min incubation on living ECs either in the absence (top left panel) or the presence (middle left panel) of 12G10 or TS2/16 (bottom left panel). To thoroughly analyze the morphology of ECM adhesion sites, g -STED confocal images were acquired close to the basal EC surface. 9EG7-labeled adhesions were then analyzed with ImageJ software (right panels) and classified, according to their shape factor (SF), into elongated (red) and round (yellow) structures. 9EG7-labeled adhesions were classified as elongated, if their SF was < 0.5, and round, if the SF was ≥ 0.5. Scale bar 20 µ m. The maximum Feret’s diameter was measured to quantify the morphological features of 9EG7 + elongated structures. Compared with control ECs incubated live with 9EG7 alone, 9EG7 + elongated structures were significantly shortened in the presence of 12G10 or TS2/16. Data are mean ± SD, n ≥ 20 cells per condition pooled from two independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Staining, Incubation, Binding Assay, Confocal Microscopy, Labeling, Software, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Low magnification confocal immunofluorescence microscopy analysis of ECs that were treated live for 10 min with either mAb 9EG7 mAb ( green ) alone or in combination with mAb 12G10 ( red ). Scale bar 100 µ m.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Representative confocal microscopy analysis of SNAKA51 + active α5 integrin (A), β3 integrin (B), and soluble rhodamine-FN (C) localization in ECs that were incubated (bottom panels) or not (top panels) for 10 min with the anti-βI domain mAb 12G10. Scale bar 20 µ m; magnification scale bar 10 µ m. (A, C) When compared with untreated control (CTL) ECs, the mFD of SNAKA51 + fibrillar adhesions or rhodamine-FN + fibrils was significantly reduced in ECs treated with mAb 12G10. Data are mean ± S.D, n ≥ 34 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (B) The incubation of cultured ECs with mAb 12G10 did not influence number, mean area, or mFD of β3 integrin + focal adhesions. Data are mean ± S.D, n ≥ 32 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Representative g -STED confocal microscopy analysis of tensin 1 localization in ECs that were incubated or not for 10 min with the anti-active β1 integrin mAb 9EG7 or mAb 12G10 or the Fab fragment of mAb 12G10 (12G10-Fab) or mAb TS2/16. Scale bar 20 µ m; magnification scale bar 10 µ m. When compared with untreated control (CTL) ECs or those treated with mAb 9EG7, the mFD of tensin 1 + fibrillar adhesions was significantly reduced in ECs treated with either mAb 12G10, 12G10-Fab or mAb TS2/16. Data are mean ± SD, n ≥ 22 cells per condition pooled from three independent experiments. The number of structures was normalized on cell area and on those in control cells. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (B) MAb 9EG7 affects the lifetime of FN–α5β1 bonds. AFM measurement of mAb 9EG7 effect on force-dependent lifetime of single bonds between a FNIII 7–10 fragment and an integrin α5β1-Fc fusion protein. Lifetime versus force plots of α5β1-Fc–functionalized Petri dish dissociating from FNIII 7–10 -coated cantilever tips in Mn 2+ either in the absence (grey) or the presence (green) of 10 µ g/ml mAb 9EG7 mAb. Data are mean ± SEM of several tens to several hundreds of measurements per point. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Selected frames from (A), (B), and (C), monitoring mAb 12G10 ( red ) binding to active β1 integrins on living: control ECs (A); ECs previously oligofected with tensin-EGFP (B); ECs pre-incubated for 10 min with mAb 9EG7 (C). (A, B, C) As expected, mAb 12G10 mAb binds active β1 integrins located within and outside fragmented adhesion sites (A), but, either when Tensin-EGFP is overexpressed (B) or upon pre-incubation with mAb 9EG7 (C), mAb 12G10 mAb localizes instead within fibrillar adhesions that remain stable over time. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Representative confocal images showing pre-bleaching, post-bleaching, and recovery on the region of interest (indicated by arrows) of tensin-EGFP–positive fibrillar adhesions in ECs treated with DMSO (as control) or with the FAK inhibitor PF-562271. Scale bar 5 µ m. Recovery rate was measured, and data were normalized by employing as reference the fluorescence intensity acquired on the same ROI before bleaching. Data were then normalized on control DMSO-treated samples. Data are mean ± SD, n ≥ 31 adhesions per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.001 ***. The effectiveness of inhibition of FAK autophosphorylation by PF-562271 was verified by Western blot analysis of EC lysates. (E) Representative confocal microscopy images of anti-active β1 mAb 12G10 ( green ) localization in ECs treated or not with the FAK inhibitor PF-562271; ECs were also stained for auto-phosphorylated FAK on tyrosine 397 (pFAK-Y397, red ). Scale bar 20 µ m; magnification scale bar 10 µ m. Measurement of mFD of 12G10 + adhesions revealed that, compared with control EC incubated with DMSO, 12G10 + adhesive structures are significantly longer in presence of FAK inhibitor PF-562271. Data are mean ± SD, n ≥ 19 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (F) Western blot analysis of FAK autophosphorylation (Y397) levels in ECs treated with or without mAb 9EG7 or mAb 12G10 for 2 or 15 min. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Binding Assay, Control, Incubation, Fluorescence, Inhibition, Western Blot, Confocal Microscopy, Staining, Adhesive

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Representative confocal microscopy analysis of the impact of mAb 12G10 on living ECs previously incubated for 10 min either with intact mAb 9EG7–Alexa Fluor 488 (9EG7) or its Fab (9EG7-Fab). Scale bar 10 µ m; magnification scale bar 10 µ m. Upon pre-incubation with intact dimeric 9EG7, but not monomeric 9EG7-Fab, mAb 12G10 localizes within fibrillar adhesions. Quantitative analysis showed that the incubation of cultured ECs with 12G10 shortens the mFD of elongated active β1 integrin + clusters only when cells are pre-incubated with monomeric 9EG7-Fab, but not intact dimeric 9EG7. Data are mean ± S.D, n ≥ 22 cells per condition pooled from three independent experiments. Statistical analysis: one-way ANOVA, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy analysis of ECs live treated with mAb 9EG7 ( green ) alone or in combination with mAb 12G10 ( blue ). The internalization of mAb 9EG7–bound active β1 integrins in EEA1 + early endosomes ( red ) was quantified by Pearson correlation coefficient (PCC). Treatment with the anti-βI domain mAb 12G10 promotes mAb 9EG7–bound active β1 integrin endocytosis. Data are mean ± SD, n ≥ 23 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. (B) Time-course analysis of the relative amounts of endocytosed 9EG7 + active β1 integrins in control (CTL; green ) versus mAb 12G10–treated ( light blue ) ECs, evaluated by internalization and capture ELISA assays. Treating living ECs with the anti-βI domain mAb 12G10 elicits a strong increase in 9EG7 + active β1 integrin endocytosis. Data are mean ± SD, of eight technical replicates per condition pooled from three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (C, D) Real-time analysis of EC haptotactic migration towards FN (xCELLigence RTCA DP system) either in the absence (CTL) or the presence of anti-active β1 integrin mAb 9EG7 alone (C, D) or mAb12G10 alone (C) or combined mAb 9EG7 and mAb 12G10 (D). Data are mean ± SD, n ≥ 14 technical replicates per condition pooled from four independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.05 * , # ; P ≤ 0.01 ** , ## ; P ≤ 0.001 *** , ### ; P ≤ 0.0001 **** , #### . (C, D) *CTL versus mAb 9EG7; # CTL versus mAb 12G10 (C); or CTL versus mAb 9EG7 + mAb 12G10 (D). Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Control, Enzyme-linked Immunosorbent Assay, Migration

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Confocal immunofluorescence microscopy analysis of ECs live treated with mAb SNAKA51 ( red ) alone or in combination with mAb 12G10 ( green ). The internalization of mAb SNAKA51–bound active α5 integrins in EEA1 + early endosomes ( blue ) was quantified by Pearson correlation coefficient. Treatment with the anti-βI domain mAb 12G10 promotes mAb SNAKA51–bound active α5 integrin endocytosis. Data are mean ± SD, n ≥ 26 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Journal: PLoS Biology

Article Title: BMPR2 acts as a gatekeeper to protect endothelial cells from increased TGFβ responses and altered cell mechanics

doi: 10.1371/journal.pbio.3000557

Figure Lengend Snippet: (A) Immunoblot showing total β1-integrin (tITGB1) levels and phosphorylation at Ser785 and Threonine 783 (left) under steady-state conditions and densitometric quantification of total or phosphorylated β1-integrin levels in indicated cell clones (right). Data are presented as mean + SD ( n = 3–4 independent experiments). (B) PLA between active β1-integrin (ITGB1 pS785 ) and ILK under steady-state growth conditions. (C) Single confocal z-planes of BMPR2 wt (basal, left), BMPR2 ΔE2 (medial, middle), and BMPR2 KO (medial, right) depicting the localization of ILK (green) or paxillin (red). Insets show zoomed-in regions. (D) Immunoblots showing protein levels of VE-Cadherin and phosphorylated VE-Cadherin in indicated ECs under steady-state conditions. (E) Single confocal z-planes of immunocytochemical staining using antibodies specific for VE-Cadherin (green) and PECAM-1 (red). Insets show zoomed-in regions. (F) Immunoblots showing levels of phosphorylated cofilin (pSer-3), pMLC (pSer-19), and total MLC in indicated ECs under steady-state culture conditions. (G) Cartoon (left) depicting the principle of CFS (particle diameter 23 μm). Elastic modulus derived from CFS of living cells (middle). Representative indentation (force versus distance) curves for the different cell lines and the hard control surface (mica; right). Data are shown as mean + SD ( n = 30). (H) Principle of QI using a sharp cantilever tip (diameter < 20 nm; left). Representative QI scans of fixed cells, focusing on CCC sites (middle). Height profiles of nucleus-to-cell junction height differences (black arrows) are indicated. Given values are expressed as the mean ± SD. Height profile measurements were taken from n ≥ 18 cells; scale bars, 10 μm; Statistical significance relative to BMPR2 wt was calculated using Kruskal-Wallis test with post hoc Dunn test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. See also and for underlying data. BMP, bone morphogenetic protein; BMPR2, BMP type-2 receptor; CCC, cell-to-cell contact; CFS, colloidal force spectroscopy; EC, endothelial cell; ILK, integrin-linked kinase; MLC, myosin light chain; PECAM-1, platelet endothelial cell adhesion molecule-1; PLA, proximity ligation assay; pMLC, phosphorylated MLC; QI, quantitative imaging; pS785, phosphorylated Serine 785; pT783, phosphorylated Threonine 783; tITGB1, total integrin beta-1; VE, vascular endothelial.

Article Snippet: Respective dilutions are indicated as follows: phosphorylated SMAD1/5, clone 41D10 (WB 1:1000; Cell Signaling Technologies); phosphorylated SMAD2, clone 138D4 (WB 1:1000; Cell Signaling Technologies); SMAD1, clone D59D7 (WB 1:1000; Cell Signaling Technologies, ChIP 5 μg/immunoprecipitation); SMAD1, AB55476 (PLA 1:200; Abcam); SMAD2 clone 86F7 (WB 1:1000; PLA 1:200; Cell Signaling Technologies); SMAD2/3 clone 18 (WB 1:1500; PLA 1:200; BD Biosciences); SMAD5 12167-1-AP (WB 1:1000; PLA 1:100; Proteintech); SMAD4 clone D3R4N (WB 1:1000; PLA 1:800; Cell Signaling Technologies); ILK clone 65–1 (WB 1:500; ICC 1:50; PLA 1:50; Santa Cruz Biotechnology); β1-integrin (CD-29) clone 18 (WB 1:1000; BD Biosciences); β1-integrin clone P5D2 (ICC 1:500; Abcam); phosphorylated β1-integrin-Ser785 AB8124 (WB 1:10000; PLA 1:500; Merck Millipore); phosphorylated β1-integrin-Tyr783 AB8125 (WB 1:10000; Merck Millipore); phosphorylated paxillin-Tyr118 (ICC 1:100; Cell Signaling Technologies); N-Cadherin clone 32 (ICC 1:250; BD Biosciences); VE-Cadherin clone D87F2 (WB 1:1000); phosphorylated VE-Cadherin-Tyr685 CP1981 (WB 1:1000; ECM Biosciences); VE-Cadherin clone BV6 (ICC 1:200; Merck Millipore); PECAM-1 (CD-31) clone 89C2 (ICC 1:3000; immunohistochemistry [IHC]: 1:250; Cell Signaling Technologies); LTBP-1 (IHC: 1:1000; the antibody was previously described [ ]); β-Catenin clone D10A8 (ICC 1:250; Cell Signaling Technologies); αSMA (IHC: 1:250 ab5694, Abcam); MLC 2 #3672 (WB 1:1000; Cell Signaling Technologies); pMLC-Ser19 #3671 (WB 1:1000; ICC 1:50; IHC: 1:50; Cell Signaling Technologies); phosphorylated cofilin-Ser3 clone 77G2 (WB 1:1000; Cell Signaling Technologies); LAP (of TGFβ-1) clone 9005 (WB 1: 1000; ICC 1:250; R&D Systems); TβR2 clone C-4 (WB: 1:500; Santa Cruz Biotechnology); BMPR2 (ICC 1:200; Cell Signaling Technologies); BMPR2 clone-18 (WB 1:1000; BD Biosciences); FN ab23750 (ICC 1:50; Abcam); GAPDH clone 14C10 (WB 1:1000; Cell Signaling Technologies); β-Actin clone AC-15 (WB 1:5000; Sigma Aldrich); myc-tag clone 9B11 (WB 1:1000; ICC 1:8000; Cell Signaling Technologies); and TGFβ-1,2,3 MAB1835 (WB 1:1000; R&D Systems).

Techniques: Western Blot, Clone Assay, Staining, Derivative Assay, Spectroscopy, Proximity Ligation Assay, Imaging