Figure 6.

TMOD3 Mediates EC Functions and Cytoskeletal Morphology

(A and B) HRMECs were transfected with small interfering RNA (siRNA) targeting TMOD3 (siTMOD3) (5 or 10 nM) or negative control siRNA (siNC) for 24 h. (A) Treatment of siTMOD3 significantly decreased expression of TMOD3 in HRMECs. Expression levels were normalized to 18S and to cells treated with siNC (n = 3 per group). (B) Quantitative analyses of MTT assays showed that HRMECs treated with siTMOD3 had significantly increased proliferative activity (n = 9–12 per condition). (C and E) HRMECs were transfected with miR-145 inhibitors (50 nM) or negative controls (Neg Ctrl) combined with siTMOD3 (5 or 10 nM) or siNC for 24 or 48 h and analyzed for tube formation (C) and cell shape (D and E). In the tube formation assay, HREMCs transfected with siTMOD3 had a significant increase in total mesh area, even in the miR-145 inhibitor co-transfected groups (C); n = 6 cells per group. Furthermore, knockdown of TMOD3 in HRMECs show a substantially increased aspect ratio (cell length/cell width) indicating elongated cell shape compared with cells treated with siNC (D); n = 60–130 cells per group. Immunocytochemical staining of F-actin (red) and TMOD3 (green) in HRMECs demonstrated decreased TMOD3 staining, longer F-actin fibers, less cytoskeleton mesh, and elongation of cell shape in the siTMOD3-treated group (E). Scale bars represent 50 μm (E). Data are presented as means ± SEM. *p ≤ 0.05; **p ≤ 0.005; ***p < 0.001.