Small‐molecule drug screens identify compounds relevant for CCM

• A. Overview of the four different screening assays used in this study. Zebrafish embryos and C. elegans are screened in 24‐well and 96‐well plates, respectively. The most promising active compounds are retested in shCCM2 HUVECs. One compound is tested for suppression of vascular lesion formation in the cerebellum of iCCM2 and iCCM3 mouse models.

• B. Overlap of rescue compounds screened in the different assays.

• C–E Examples of rescue of cardiovascular defects of the zebrafish ccm2^m201 mutant. Inverted images of confocal z‐scan projections of the 46 hpf head region and heart (endocardium) of wild‐type (WT) and ccm2^m201 mutant zebrafish embryos carrying the endothelial Tg(kdrl:GFP)^s843 reporter transgene. Embryos are untreated (C) or treated between 17 and 48 hpf with 10 μM of the Lck inhibitor C8863 (D) or with 10 μM of the ERK5 inhibitor XMD8‐92 (E). Both compounds resulted in a reduction in heart size and narrowing of the heart tube at the atrioventricular canal (arrowheads). Scale bar is 100 μm.

Indirubin‐3‐monoxime (IR3mo) rescues the CCM phenotype in zebrafish and HUVEC models

• A–D Treatment with 5 μM indirubin‐3‐monoxime (IR3mo) rescues the embryonic zebrafish ccm2^m201 mutant ballooning heart phenotype. Shown are images of confocal z‐scan projections of wild‐type (WT) (A, C) and ccm2^m201 mutant (B, D) zebrafish embryonic hearts at 48 hpf expressing the endothelial reporter Tg(kdrl:GFP)^s843 (cyan) and counter‐stained for Alcam (magenta, labeling the myocardium). Scale bar is 50 μm.

• E–H Treatment with 10 μM IR3mo for 48 h restores the wild‐type ACTIN‐adhesive phenotype in KRIT1‐depleted HUVECs. Shown are confocal images of HUVECs with labeled VE‐cadherin (green) and F‐ACTIN (red). Control siRNA‐silenced (E, G) and KRIT1 siRNA‐silenced (F, H) HUVECs were not treated (E, F) or treated (G, H) with IR3mo. Scale bar is 10 μm.

• I–K Treatment with IR3mo reduces phosphorylation of ERK (pERK) without affecting overall ERK protein levels. Shown in (I) are representative Western blots of KRIT1‐silenced HUVECs lysates treated or not with 10 μM of IR3mo during transfection. ERK5 (J) and phosphorylated ERK5 protein levels (K) were measured relative to ACTIN protein levels based on three biological replicates with two technical replicates each (n = 3). Statistical analyses were performed using two‐way ANOVA followed by Tukey's multiple comparisons test; error bars are SD. ****P < 0.0001; ns, not significant.

• L. Treatment with IR3mo rescues the elevated KLF2 levels of KRIT1‐silenced HUVECs. RT‐qPCRs were performed to measure KLF2 levels, on three biological replicates (n = 3). Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons test; error bars are SD. ***P < 0.001; ns, not significant.

• M. Treatment with IR3mo rescues the elevated klf2a levels of ccm2^m201 mutant zebrafish embryos at 48 hpf. RT‐qPCRs were performed to measure klf2a levels, on six biological replicates (n = 6). Statistical analyses were performed using one‐way ANOVA followed by Tukey's multiple comparisons test; error bars are SD. *P < 0.05; ns, not significant.

Indirubin-3-monoxime (IR3mo) rescues the CCM phenotype in krit1^ty219c mutant zebrafish and CCM2- and CCM3-depleted HUVECs.

Treatment with 5 lM IR3mo rescues the embryonic zebrafish krit1ty219c mutant ballooning heart phenotype. Shown are images of confocal z-scan projections of wild-type (WT) (A, C) and krit1ty219 mutant (B, D) zebrafish embryonic hearts at 48 hpf expressing the endothelial reporter Tg(kdrl:GFP)s843 (green) and counterstained for ACTIN (red). Scale bar is 50 lm.

Treatment with 10 lM IR3mo for 48 h rescued the CCM phenotype in CCM2- or CCM3-depleted HUVECs. Shown are confocal images of HUVECs with labeled VE-cadherin (green) and F-ACTIN (red). Control siRNA-silenced (E, F), CCM2 siRNA-silenced (G, H), or CCM3 siRNA-silenced (I, J) HUVECs untreated (E, G, I) or treated (F, H, J) with IR3mo. Scale bar is 10 lm.