Figure 6.
The Rho/SLK axis downstream of β3-integrin controls ERM activation and sealing zone formation. (A) Effect of calyculin and staurosporine treatment on ERM activation (P-ERM), used as positive and negative control for western blot analysis of ERM activation, respectively. 6-day hOCs were treated or not (CTL) with calyculin and staurosporine. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. (B) RhoA inhibition reduces ERM activation. 6-day hOCs were treated or not (CTL) with TATC3, targeting the RhoGTPases RhoA. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 6, means ± SDs are shown. (C) SLK suppression reduces ERM activation. hOCs were treated with non-targeting siRNA (siCTL) or siRNA targeting SLK kinase (siSLK). Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 7, means ± SDs are shown. (D) β3-integrin suppression favors ERM activation. hOCs were treated with nontargeting siRNA (siCTL) or siRNA targeting β3-integrin (si β3-integrin). Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 7, SDs are shown. Predicted molecular weights are indicated (A–D). (E and F) Effect of SLK and β3-integrin depletion on the formation of sealing zones in hOCs. (E) Representative microscopy images of sealing zones visualized by F-actin staining (phalloidin: white in upper panels and colored-coded intensity in lower panels). Scale bars, 20 and 5 µm. (F) Quantification of sealing zone thickness (n = 2 donors, 15–20 cells/condition and 3 locations/SZ). (G) Schematics showing the proposed Rho/SLK axis downstream of β3-integrin for ERM activation. Statistical analyses: Multiple comparison tests (A) Friedman and then Dunn’s, and (F) Kruskal–Wallis and then Dunn’s. ****P ≤ 0.0001. Source data are available for this figure: SourceData F6.

The Rho/SLK axis downstream of β3-integrin controls ERM activation and sealing zone formation. (A) Effect of calyculin and staurosporine treatment on ERM activation (P-ERM), used as positive and negative control for western blot analysis of ERM activation, respectively. 6-day hOCs were treated or not (CTL) with calyculin and staurosporine. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. (B) RhoA inhibition reduces ERM activation. 6-day hOCs were treated or not (CTL) with TATC3, targeting the RhoGTPases RhoA. Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 6, means ± SDs are shown. (C) SLK suppression reduces ERM activation. hOCs were treated with non-targeting siRNA (siCTL) or siRNA targeting SLK kinase (siSLK). Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 7, means ± SDs are shown. (D) β3-integrin suppression favors ERM activation. hOCs were treated with nontargeting siRNA (siCTL) or siRNA targeting β3-integrin (si β3-integrin). Representative western blot analysis (left) and quantification of P-ERM signal normalized to actin (right). Each circle represents a single donor, n = 7, SDs are shown. Predicted molecular weights are indicated (A–D). (E and F) Effect of SLK and β3-integrin depletion on the formation of sealing zones in hOCs. (E) Representative microscopy images of sealing zones visualized by F-actin staining (phalloidin: white in upper panels and colored-coded intensity in lower panels). Scale bars, 20 and 5 µm. (F) Quantification of sealing zone thickness (n = 2 donors, 15–20 cells/condition and 3 locations/SZ). (G) Schematics showing the proposed Rho/SLK axis downstream of β3-integrin for ERM activation. Statistical analyses: Multiple comparison tests (A) Friedman and then Dunn’s, and (F) Kruskal–Wallis and then Dunn’s. ****P ≤ 0.0001. Source data are available for this figure: SourceData F6.

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