Figure 3.

TMEM215 was required for EC survival. (a, b) HUVECs were transfected with Si‐Ctrl, Si‐215‐1, or Si‐215‐2, and mRNA level of TMEM215 was determined by qRT‐PCR. (b) HUVECs expressing a Flag‐tagged TMEM215 were transfected with Si‐Ctrl, Si‐215‐1, or Si‐215‐2, and protein level of TMEM215‐Flag was determined by western blot analysis using anti‐Flag. (c) HUVECs were transfected with Si‐Ctrl or Si‐215‐1, and cell viability was evaluated by MTT assay (n = 6). (d) HUVECs were transfected with Si‐Ctrl or Si‐215‐1, and cell proliferation was evaluated by EdU incorporation assay (n = 7). (e) HUVECs were transfected with Si‐Ctrl, Si‐215‐1, or Si‐215‐2, and cell apoptosis was evaluated by TUNEL (n = 5). (f) HUVECs were transfected with Si‐Ctrl or Si‐215‐1, and cells were observed under TEM. (g, h) HUVECs were transfected with Si‐Ctrl, Si‐215‐,1 or Si‐215‐2, and stained by Annexin V and PI followed by FACS analysis. Percentage of dead cells was determined (n = 5). (i) HUVECs were transfected with Si‐Ctrl or Si‐215‐1, and LDH in the culture medium was determined (n = 5). Bar = means ± SD, *p < 0.05, **p < 0.01, and ***p < 0.001. EC: endothelial cell; EdU: 5‐ethynyl‐2′‐deoxyuridine; FACS: fluorescence‐activated cell sorting; HUVEC: human umbilical vein endothelial cells; LDH: lactate dehydrogenase; N.S.: not significant; mRNA: messenger RNA; MTT: 3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide; PI: propidium iodide; qRT‐PCR: quantitative reverse transcription‐polymerase chain reaction; SD: standard deviation; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling [Color figure can be viewed at wileyonlinelibrary.com]