Fig. 3

Fig. 3

A The diagram showing the molecules related to nicotinamide. Nicotinamide (Nam) and niacin can be transformed to NAD⁺ by different enzymes. Nicotinamide blocks the activation of these NAD⁺ consuming enzymes such as SIRT and PARP, and it also inhibits the activity of ROCK and CSNK1. B Analyses of mRNA expression levels of NKX2-5 and TNNT2 at day 10 of differentiation with the indicated treatments. hESC H1 cells were treated with IWP-2 3 μM from day 2 to day 5, Nicotinamide (Nam) 20 mM, Niacin 10 mM, EX527 (SIRT1i) 10 μM, AZD2281 (PARPi) 100 nM, Y-27632 (ROCKi) 10 μM or D4476 (CSNK1i) 10 μM from day 1 to day 5. C TREEspot^TM interaction maps of nicotinamide. % Control indicates the binding efficiency between the substrate and specific kinase, and lower numbers indicate stronger inhibition. Kinase hits (% Control < 35) are labeled with red circles, and larger circles indicate the kinases with lower % Control. D The target kinases with % Control < 20 under 3 mM nicotinamide treatment from the DiscoverX KINOMEscan service. Lower numbers of % Control indicates higher-affinity binding. E The influence of nicotinamide on P38δ activity in vitro measured by the bioluminescent kinase assay (n = 3 technical replicates). The red circle indicates different doses of nicotinamide (Nam), and the black square indicates SB202190 (P38i) 10 μM. The data shown are representative of three independent experiments. F Analyses of mRNA expression levels of NKX2-5 and TNNT2 on day 10 of differentiation treated with Nicotinamide (Nam) 20 mM, SB202190 (P38i-1) 10 μM, SB203580 (P38i-2) 10 μM, or BIX02189 (MEK5i) 5 μM from day 1 to day 5. G Representative flow cytometric analyses showing the percentage of TNNT2-positive cells after 13 days of differentiation induced by SB202190 (P38i) or nicotinamide (Nam). Results are representative of three independent experiments. Data shown are normalized to control, and presented as mean ± SD of three independent experiments (*p < 0.05 compared with Control).