Fig. 7

A Effect of nicotinamide on the development of The Tg(Fli1:EGFP) transgenic zebrafish. Nicotinamide was applied between 18 and 24 h post fertilization. The images were taken after 3 days of treatment. Top, the bright field images of zebrafishes; Bottom, the fluorescence images. Scale bar, 200 μm. B The enlarged images showing the intersegmental vessels (ISV) and subintestinal veins (SIV) of zebrafishes. Scale bar, 100 μm. C Percentage of zebrafish with pericardial edema, impaired ISV, or impaired SIV (n = 22 in control group, and n = 26 in nicotinamide group). D The schematic diagram showing the protocol of endothelial and smooth muscle differentiation. VEGF 50 ng/mL was added from day 2 to day 5 in the absence or presence of 10 mM nicotinamide. E Analyses of mRNA expression levels of PECAM, CDH5, PDGFRβ, and ACTA2 by qPCR at day 9 of differentiation. Black, Control; green, VEGF 50 ng/mL; blue, Nicotinamide 10 mM; red, VEGF 50 ng/mL and nicotinamide 10 mM. Data shown are normalized to control and represent three independent experiments (*p < 0.05 compared with control, #p < 0.05 compared with VEGF). F The indicated inhibitors were added to analyze the mechanism of nicotinamide in VEGF-induced differentiation. The mRNA levels of PDGFRβ and ACTA2 were measured by q-PCR at day 9 of differentiation. The following treatments were added from day 2 to day 5 together with VEGF (50 ng/mL): Nicotinamide (Nam,10 mM), P38 inhibitor SB202190 (P38i, 10 μM), ROCK inhibitor Y-27632 (ROCKi, 10 μM), CSNK1 inhibitor D4476 (CSNK1i, 10 μM), MEK5 inhibitor BIX02189 (MEK5i, 5 μM), PARP inhibitor AZD2281 (PARPi, 100 nM), SIRT1 inhibitor EX527 (SIRT1i, 10 μM), or Niacin (10 mM). Data shown are normalized to control and represent mean ± SD of three independent experiments (#p < 0.05 compared with VEGF).