Fig. 5

Silencing of MEG3 inhibited apoptosis in PCOS rat ovarian granulosa cells. A Experimental strategy for the inhibition of ovarian MEG3 expression by viral injection; B The JC-1 method was used to detect the early stage of apoptosis in PCOS rat ovarian granulosa cells after MEG3 downregulation (scale bar = 100 μm):at high mitochondrial membrane potential, which can produce red fluorescence, at low mitochondrial membrane potential, which produces green fluorescence, and the blue fluorescent DAPI dye labels the nucleus, and Merge is a superimposed image showing the three fluorescent signals; C The ratio of JC-1 aggregate (red) to monomer (green) intensity in myotubes, Cell experiments were independently repeated three times; D cleaved caspase-3, caspase-3, Bax and Bcl-2 protein bands in PCOS rat ovarian granulosa cells after MEG3 downregulation; E Western blotting to detect the Bcl-2/Bax ratio in PCOS rat granulosa cells after MEG3 downregulation in granulosa cells; F Western blotting to detect cleaved caspase-3/caspase-3 protein expression in PCOS rat granulosa cells after MEG3 down regulation; G Detection of caspase-3 by RT‒qPCR in ovarian granulosa cells of PCOS rats; H Detection of Bax by RT‒qPCR in ovarian granulosa cells of PCOS rats; I Detection of Bcl-2 by RT‒qPCR in ovarian granulosa cells of PCOS rats; J Apoptosis of PCOS rat ovarian granulosa cells after MEG3 downregulation was detected by the TUNEL method (scale bar = 100 μm); green fluorescent FITC dye labelled apoptotic cells, blue fluorescent DAPI dye labelled nuclei, and Merge is a superimposed image showing both fluorescent signals; K fluorescence values of PCOS rat ovarian granulosa cells after MEG3 downregulation indicating apoptosis. D-K, N = 6. These data are expressed as the mean ± standard deviation, **P < 0.01 or ***P < 0.0001 compared to the normal group, ^##P < 0.01 or ^###P < 0.0001 compared to the sh-NC group and were statistically analysis using one-way ANOVA and Tukey’s post hoc test. 1: Normal group, 2: PCOS group, 3:sh-MEG3 group, 4:sh-NC group