Additional file 1 of Nicotinamide promotes pancreatic differentiation through the dual inhibition of CK1 and ROCK kinases in human embryonic stem cells

Additional file 1: Supplemental Figure 1. Enhancement of pancreatic progenitor induction by nicotinamide depends on kinase inhibition, but not on PARP and SIRT regulation. a Nicotinamide`s molecular targets screened via KINOMEscan™ confirmed that nicotinamide can directly bind and inhibit CK1 and ROCK; b RT-qPCR analysis for mRNA levels of NKX6.1 and PDX1 in pancreatic progenitors on day 13 of differentiation from H9, treated with nicotinamide, D4476 and Y27632 (n = 3), *p 3), *p 3), *p 3), *p < 0.05; c Flow cytometry analysis to test the effect of nicotinamide, nicotinamide+D4476 and nicotinamide+Y27632 on the generation of PDX1+/NKX6.1+ cells (n = 3). Supplemental Figure 5. Global gene expression of pancreatic progenitors under different treatments. a Heatmap and hierarchical clustering of representative genes of delta, epsilon, pancreatic polypeptide (PP), Acinar and ductal cells under Mock, D4476, Y27632 and nicotinamide conditions compared to hESCs cultured in E8. Supplemental Figure 6. Pancreatic progenitors under Mock, nicotinamide, D4476 and Y27632 treatment can be further induced into insulin positive β-like cells. a Differentiation strategy from pancreatic progenitors to β-like cells; b Immunostaining for insulin expression in β-like cells generated from pancreatic progenitors under Mock, nicotinamide, D4476 and Y27632 condition, Scale bar 400 μM and 100 μm.附加文件1：补充图1。烟酰胺对胰腺祖细胞诱导的增强效应依赖于激酶抑制，而非PARP与SIRT调控。a 经KINOMEscan™筛选验证的烟酰胺分子靶点实验证实，烟酰胺可直接结合并抑制CK1与ROCK；b 对经H9细胞分化13天得到的胰腺祖细胞，分别用烟酰胺、D4476、Y27632处理后，通过RT-qPCR（实时荧光定量聚合酶链式反应）分析NKX6.1与PDX1的mRNA水平（n=3），*p<0.05；c 流式细胞术分析烟酰胺、烟酰胺联合D4476、烟酰胺联合Y27632对PDX1+/NKX6.1+细胞生成的影响（n=3）。 补充图5：不同处理条件下胰腺祖细胞的全局基因表达谱。a 相较于在E8培养基中培养的人类胚胎干细胞（hESCs），Mock组、D4476组、Y27632组及烟酰胺组处理条件下，δ细胞、ε细胞、胰多肽（PP）细胞、腺泡细胞及导管细胞的代表性基因的热图与层级聚类分析。 补充图6：经Mock组、烟酰胺、D4476及Y27632处理得到的胰腺祖细胞可进一步诱导为胰岛素阳性β样细胞。a 从胰腺祖细胞向β样细胞分化的策略；b 对上述各组胰腺祖细胞诱导得到的β样细胞进行胰岛素表达免疫荧光染色，标尺分别为400μm与100μm。