Fig. 8
aPC epigenetically inhibits glucose-induced sustained p66Shc expression. a Representative reverse-transcriptase-PCR gel images (out of three independent repeat experiments, top) showing methylated (M) and unmethylated (U) p66Shc promoter DNA in BMDMs with treatment as indicated and dot plot summarizing the ratio of methylated to unmethylated p66Shc promoter DNA (bottom). b Glucose-induced sustained p66Shc mRNA expression is prevented by aPC (20 nM) in BMDMs cells. Representative RT-PCR gel image (out of five independent repeat experiments, top; β-actin: loading control) and dot plot summarizing data (bottom). c Representative immunoblot images (out of 3 independent repeat experiments with two technical replicates each) for p66Shc and DNMT1 expression in BMDMs with treatment as indicated (left panel; GAPDH: loading control) and dot plot summarizing data (middle and right panels). d dot plot summarizing DNMT activity in BMDMs with treatment as indicated. NG: normal glucose (5 mM glucose plus 20 mM mannitol, 48 h), HG: high glucose (25 mM, 48 h). HG-NG: HG (24 h) followed by NG (24 h) condition; HG-NG-aPC: HG-NG conditions with additional exposure to aPC (20 nM) during the last 24 h); HG-NG-Aza: HG-NG conditions with additional exposure to the DNMT-inhibitor 5-azacytidine (Aza, 5 µM) during the last 24 h; HG-NG-aPC-Aza: HG-NG conditions with additional and concomitant exposure to aPC (20 nM) and 5-azacytidine (5 µM) during the last 24 h. Data shown represent mean ± SEM of at least three independent experiments each with at least two technical replicates (a–d); **P < 0.01; one-way ANOVA with Bonferroni-adjusted post hoc comparison of HG and HG-NG versus NG, HG-NG-aPC versus HG-NG (a, b, d), and HG-NG, HG-NG-Aza, HG-NG-aPC-Aza versus DM-NG-aPC). Uncropped reverse-transcriptase-PCR gel images for Fig. 8a, b and immunoblots for Fig. 8c are provided in Supplementary Figure 23
