Figure 6. NEUROG3 Is Phosphorylated by G₁-S Cyclin-Dependent Kinases.

(A) Anti-FLAG western blot of HeLa cell lysates expressing indicated NEUROG3 variants were incubated with nocodazole alone or in combination with BIX02189, LY2228820, SP600125, or SB216763.

(B) Anti-FLAG western blot on HeLa cell lysates expressing FLAG-tagged WT-NEUROG3 were either untreated, incubated with nocodazole alone, or incubated with both nocodazole and roscovitine.

(C) Mouse pancreatic explants were cultured for 24 hr starting at E11.5 with Cdk2 and 4/6 inhibitors (CDKi) and the number of Neurog3⁺ cells/Sox9⁺ cells and the total Sox9⁺ cells were quantified by immunofluorescence staining. n = 5. **p < 0.01 by unpaired t test.

(D) Stage-6 endocrine progenitor cells derived from a human iPSCs were treated for 24 hr with CDKi and NEUROG3 protein was quantified by western blot. n = 3 wells. **p < 0.01 by unpaired t test.

(E) GFP⁺ cells were quantified using a NEUROG3-2A-eGFP hESCs (N5-5) differentiated to stage 6 prior to 24-hr CDKi treatment. n = 9 wells from two differentiations. *p < 0.05 by Mann-Whitney U test.

(F) NEUROG3 expression in FACS purified GFP⁺ N5-5 cells following CDKi treatment at stage 6. n = 3 wells from two differentiations.

(G) CDKi treatment increased expression of NEUROD1 in FACS purified GFP⁺ cells. n = 3 wells from two differentiations. *p < 0.05 by one-way ANOVA and Tukey post hoc test.

(H) CDKi treatment increased expression of PDX1 in FACS purified GFP⁺ cells. n = 3 wells from two differentiations. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Tukey post hoc test.

(I) CDKi treatment of stage-6 endocrine progenitors, derived from the FUCCI-3 hESC line, increased the proportion of cells in G₁ (red) and decreased the proportion of cells in S-G₂-M (green). n = 5 wells from two differentiations. ****p < 0.0001 by one-way ANOVA and Tukey post hoc test.

(J) CDKi treatment of stage-6 endocrine progenitors derived from FUCCI-3 hESC line significantly increased expression of NEUROG3 in G₁ (red) cells. n = 4 wells from two differentiations. *p < 0.05, **p < 0.01 by one-way ANOVA and Tukey post hoc test.

(K) Expression of NEUROD1 was upregulated in G₁ (red) cells compared with S-G₂-M (green). n = 5 wells from two differentiations.

(L) CDKi treatment of stage-6 endocrine progenitors derived from FUCCI-3 hESC line significantly increased expression of PDX1 in G₁ (red) cells. n = 5 wells from two differentiations. **p < 0.01 by one-way ANOVA and Tukey post hoc test.

Data are presented as mean ± SEM. See also Figure S5.