Fig. 2: miR-494 drives pathological and physiological endothelial senescence by disrupting DNA repair.

a SA-β-gal assay in early-passage HUVECs transfected with a miR-negative control or miR-494 mimic. Bars show % (mean + SEM) of β-gal-positive cells for at least 100 cells analyzed 48 h post transfection. b SA-β-gal assay at 48 h post radiation (10 Gy) of early-passage HUVECs transfected with either an inhibitor control or inhibitor miR-494. Bars show % (mean + SEM) of β-gal-positive cells for at least 100 cells analyzed. c, d SA-β-gal assay (c) and caspase-3/7 activity (d) in senescent (psg 25) HUVECs transfected with either an inhibitor control or inhibitors of miR-494 at 48 h post transfection. Bars show mean + SEM. e γH2AX staining in HUVECs 48 h after transfection with a miR-negative control or miR-494 mimic. Scale bar represents 50 µm. Bars depict mean γH2AX area per nuclear area + SEM of 3–4 technical replicates/group. f HUVECs were transfected with a miR-negative control or miR-494 mimic. After 48 h, telomerase activity was assayed using a Telo-Tagg assay. Bars depict % mean + SEM. g Representative western blot of senescence marker p21 and pRB in HUVECs transfected as in (f). h HUVECs were transfected with either a miR-negative control or miR-494 mimic. After 48 h, cells were fixed and stained with phalloidin (red) and DAPI (blue) as indicated. Scale bar represents 50 µm. Bars depict % mean cellular area + SEM. *P < 0.05; two-tailed Student’s T-test