NAD+ is critical for maintaining acetyl-CoA and H3K27ac in embryonic stem cells by Sirt1-dependent deacetylation of AceCS1

P < 0.001, **P < 0.005, *P < 0.05.(C) Western blot analysis of Tom20 in WT and Plin2 -/-mESCs (KO1# and KO2#) treated with vehicle or NMN.The experiments were repeated independently twice with similar results.(D) qPCR analysis of mtDNA copy numbers in WT and Plin2 -/-mESCs (KO1# and KO2#) treated with vehicle or NMN.Data are mean ± SD, n = 3 biological replicates.*P < 0.05.(E) Relative Sirt1 deacetylase activity in WT and Plin2 -/-mESCs (KO1# and KO2#).Data are mean ± SD, n = 3 biological replicates.*P < 0.05.(F) Western blot analysis of acetylation of AceCS1 in WT and Plin2 -/-mESCs (KO1# and KO2#).Data are mean ± SD, n = 3 biological replicates.**P < 0.005.(G) Relative Sirt1 deacetylase activity in WT and Plin2 -/-mESCs treated with vehicle or NMN.Data are mean ± SD, n = 3 biological replicates from KO1# and KO2#.***P < 0.001, ns: none significant.(H) Western blot analysis of acetylation of AceCS1 in WT and Plin2 -/-mESCs treated with vehicle or NMN.The experiments were repeated independently twice with similar results.(I) qRT-PCR analysis of expression of pluripotency genes (Oct4 and Nanog) in Plin2 -/- mESCs transfected with shRNA against Sirt1 on day 3 of differentiation in the presence of vehicle or NMN.Data are mean ± SD, n = 3 biological replicates.Two-tailed unpaired t-tests.***P < .001,ns: none significant.(J) Acetyl-CoA levels in WT and Plin2 -/-mESCs in the presence of acetate, NMN or acetate plus NMN.Data are mean ± SD, n = 2 biological replicates from KO1# and KO2#.**P < 0.005, *P < 0.05.(K) Graphical summary.