Fig. 4

miR-941 inhibition intensifies OGDR-induced programmed necrosis in human endometrial cells. The stable T-HESC cells with the pre-microRNA-941 anti-sense lentivirus (“antagomiR-941”, two lines, “L1/L2”) or microRNA anti-sense control lentivirus (“antaC”) were subjected to OGD exposure for 4 h, followed by re-oxygenation (“OGDR”) for applied time periods, ROS production (superoxide contents, a), mitochondrial CypD-ANT1-p53 association (tested by mito-IP assay, b) as well as mitochondrial depolarization (JC-1 green fluorescence accumulation, c) and cytochrome C release (d, testing cytosol proteins) were tested; Cell necrosis was tested by medium LDH release assay (e). The primary human endometrial cells were infected with antagomiR-941 or antaC for 48 h, afterwards cells were subjected to the same OGDR stimulation and cultured for applied time periods, ROS production (f), mitochondrial depolarization (g) and cytosol cytochrome C release (h) were tested similarly, with cell survival and necrosis tested by CCK-8 (i) and LDH release (j) assays, respectively. For the mito-IP assay, CypD-bound ANT1 and p53 were quantified (b), with expression of CypD, ANT1 and p53 tested in the “Input” controls (b). For the cytochrome C release assay, relative cytosol cytochrome C level was quantified (d and h). Data were presented as mean ± SD (n = 5), and results were normalized. * P < 0.05 vs. “Mock” treatment in “antaC” cells. ^#P < 0.05 vs. OGDR treatment in “antaC” cells. Experiments in this figure were repeated five times with similar results obtained