Fig. 5: Cav1 silencing attenuates CCM3 loss-augmented Tie2 signaling and lumen enlargement in in vitro models.

HBMVECs were transfected with indicated siRNAs for 72 h. a Co-immunofluorescence staining with Tie2 and Cav1. Arrows and arrowheads indicate membrane and intracellular Tie2/Cav1, respectively. b, c Cav1 co-silencing attenuates Tie2 expression. Western blotting for Cav1-Tie2 signaling. Relative protein levels and p-Tie2/Tie2 ratios were quantified by taking Ctrl siRNA as 1.0. n = 3. d, e Biotinylation assay for cell surface and endocytic Tie2. Aliquot of input (1/5) was loaded as controls. Relative surface and endocytic Tie2 were quantified by taking Ctrl siRNA as 1.0. n = 3. f, g Tie2 stability was determined by cycloheximide (CHX) treatment followed by western blot with respective antibodies. The samples were derived from the same experiment and that gels/blots were processed in parallel. Relative protein levels were quantified by taking Ctrl siRNA as 1.0. n = 3. h Immunofluorescence staining with Tie2 and Lamp1. Arrows and arrowheads membrane-localized and lysosomal Tie2, respectively. High-power images of Tie2/Lamp1 co-localization are shown at bottom. i, j Adherens junctions (AJ) and tight junctions (TJ) were visualized by immunostaining, and percentages of disrupted (discontinuous) junctions are quantified in panel (j) (10 microscope fields in each group), n = 3. k Barrier function of ECs cultured on fibronectin-coated ECIS was assessed for transendothelial electric resistance (TEER; expressed as Ohms multiplied by cm²). n = 12 wells per group. l, m Organotypic angiogenesis assay in the absence or presence of Tie2 inhibitor Rebastinib (1 μM). EC sprouts and lumens were visualized by VE-cadherin and extracellular matrix collagen-IV staining. Arrows and arrowheads indicate normal lumen and dilated lumen, respectively. Number of branch points, mean lumen diameter, and lumen areas are quantified (m). All experiments were repeated at least three times. n = 10. Data are means ± SEM. P values are indicated, one-way ANOVA followed by Tukey’s multiple comparisons test (b, c, d, e, j, k, m); two-way ANOVA followed by Tukey’s multiple comparisons test (g). Scale bar: 20 μm (a, h, i); 100 μm (l). Source data are provided as a Source data file.