Fig 1: Effect of TET2 Acetylation on AA activity.a CMK cells were treated for 4 h with 0, 2.5 or 5 µM of TSA, nuclear protein fractions were extracted and levels of TET2 protein were detected by western blot. b CMK cells were treated for 12 h with 0, 1.25, 2.5 or 5 µM of TSA, in the presence or absence of 100 µM AA. Total genomic DNA was harvested and 5hmC/5mC was determined by dot blot analysis. TSA: Trichostatin A (Cayman, Item No. 89730). c CMK cells were treated with increasing concentrations of SRT1720 (Sirtuins activator, Cayman, Catalog #10011020) in the presence or absence of 100 µM AA for 12 h and 5hmC/5mC was determined by dot blot analysis. d Sirtinol, a known sirtuin inhibitor prevents AA mediated TET activity. CMK cells were treated with increasing concentrations (0, 100, and 200 µM) of sirtinol (Selleckchem, Catalog #S2804) in the presence or absence of 100 µM AA for 12 h and 5hmC was determined by dot blot analysis. e Human myeloid leukemia cell lines CMK, HEL or SIG-M5 with TET2 wild type, heterozygous or homozygous mutation respectively were treated with SRT1720, and cell survival was monitored for 72 h and plotted. f–g Sirtuin activation amplifies AA induced TET activity resulting in cell death. CMK cells were treated with increasing concentration of SRT1720 in the presence or absence of 100 µM AA (f) or increasing concentration of AA with 2 µM of SRT1720 (g) and cell survival was monitored at 72 h post treatment. h–i TET2+/- mononuclear cells isolated from MN patient BMs treated with 100 µM of AA or in combination with either 5 µM of TSA or SRT1720 for indicated time. h 5hmC/5mC at 24. i Cell survival at 72 h. Data are shown as mean ± SEM of triplicate and are representative of at least two independent experiments; statistical significance (p values) from two tailed t test are indicated; *p < 0.05, **p < 0.01, ns not significant.
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