Figure 4

Enhanced hypoxia-induced apoptosis after miR-16-5-p mimic-transfection. (a) Representative images of TUNEL-positive cells [green]; nuclei were stained with 4ʹ, 6-diamidino-2-phenylindole (DAPI) [blue]. NRVMs were labeled with an antibody against cardiac α-sarcomeric actinin (SA) [white]. Left side; NRVMs under hypoxia. Right side; NRVMs under hypoxia with miR-16-5p mimic transfection. Upper panel; TUNEL-positive cell. Middle panel; merged image of TUNEL, DAPI. Lower panel; merged image of TUNEL, DAPI, and SA image. Yellow arrows; representative TUNEL-positive cells. Scale bar = 50 μm. (b) The ratio of TUNEL-positive apoptotic NRVMs (normoxia; n = 6, hypoxia; n = 8). Left side; normoxia with (left bar) and without (right bar) miR-16-5p mimic transfection. Right side; hypoxia with (left bar) and without (right bar) miR-16-5p mimic transfection. The percentage of apoptotic NRVMs in all NRVMs, as evaluated by the TUNEL assay. Quantitative (q) RT-PCR analysis of the mRNA expression in NRVMs. The expression of p53 (c) and Caspase-3 (d) in NRVMs (All; n = 7) transfected with miR-16-5p mimic was also evaluated. Left bar; hypoxia. Right bar; hypoxia with miR-16-5p mimic transfection. Western blotting (e,g) and analysis of the protein expression of Bax (f), and cleaved caspase-3 (h) in NRVMs (All; n = 6) transfected with miR-16-5p mimic was also evaluated. Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p mimic transfection.