fig6

Figure 6. The ability of Nobiletin to rescue autophagy and cell death is lost in the presence of siROR. (A) ROR elements in the promoters of autophagy target genes are underlined and highlighted in blue. (B) Western blot analysis of RORα protein expression in cardiac myocytes under normoxia (NMX) and hypoxia (HPX) in the presence and absence of Nob. Histogram represents quantitative data for the western blot, relative to α sarcomeric Actin as a loading control. (C) Western blot analysis of Beclin, and LC3 protein expression in cardiac myocytes under normoxia (NMX) and hypoxia (HPX) in the presence and absence of Nob and siRORα. Histogram represents quantitative data for the western blot, relative to α sarcomeric Actin as a loading control. (D) Upper panel, epifluorescence microscopy of cardiac myocytes expressing autophagy reporter GFP-LC3 (green puncta) with or without Chloroquine (CQ) to assess autophagic flux under normoxia (NMX) and hypoxia (HPX) in the presence and absence of Nob and siRORα, bar: 10 μm, magnified regions are depicted by the yellow boxes. Bottom panel, analysis of conditions tested on upper panel. (E) Cell viability assessed with vital dyes calcein-AM (green; live cells) and ethidium-homodimer-1 (red; dead cells), bar: 100 μm. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01, n = 3-4 independent myocyte isolations, counting > 200 cells for each condition tested.