CCM1/2 proteins control RhoA-dependent actin cytoskeleton organization by regulating β1 integrin activation. (A) Flow cytofluorometry using 9EG7 shows an increase in β1 integrin activation upon silencing of CCM1, CCM2, or ICAP-1. Error bars are means ± SEM (n = 4). (B) HUVECs depleted in ICAP-1, CCM1, or CCM2 spread for 1 h on low density of FN displayed more and larger β1 integrin containing focal adhesions (stained with 9EG7 antibody) localized all over their ventral face. Bar, 5 µm. (C) Quantification of the percentage of cells displaying central plaques. Error bars are means ± SEM (n = 3). (D and E) Quantification of RhoA activation upon ICAP-1, CCM1, or CCM2 depletion alone (D) or with additional β1 integrin silencing (E) by RhoGTP pull-downs. Error bars are means ± SEM (n = 3). RhoG-LISA measurements show that the level of RhoAGTP returned to that of CT upon ICAP-1 or CCM1/2 depletion in absence of β1 integrin (see Fig. S3 B). (F) Transversal actin bundles were observed in elongated HUVECs upon depletion of ICAP-1, CCM1, or CCM2 spread on FN for 4 h. Additional siRNA depletion of β1 but not of β3 integrin (Fig. S3 A) abolished their formation. (G) As sparse cells, confluent HUVECs displayed transversal actin stress fibers and their junctional VE-cadherin and β-catenin stainings appeared thinner and discontinuous (see Fig. S4). Transversal actin fibers were abolished upon additional depletion in β1 but not in β3 integrin. (H) Quantification of the percentage of sparse cells with transversal actin bundles in the absence or presence of additional β1 or β3 integrin depletion. Error bars are means ± SEM (n = 3). *, P < 0.05; **, P < 0.005; ***, P < 0.0005.