Fig. 6

Inhibition of GAPDH expression and/or crotonylation impacts hESC differentiation into the endodermal lineage. A shRNA knockdown (KD) of GAPDH (shGAPDH) in H9-LTR7 GFP cells was assessed by RT-qPCR analysis. shNC, non-targeting shRNA. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. B KD efficiency was determined by western blotting using the indicated antibodies. C GAPDH activity was assessed as described above. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. **p < 0.01. D RT-qPCR analysis of the expression of the indicated marker genes in control and KD cells. OCT4 and NANOG, pluripotency markers. GATA6 and SOX17, endoderm markers. MIXL1 and TBXT, mesoderm markers. SOX1 and NESTIN, ectoderm markers. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01. E GAPDH KD reporter cells were cultured in 10 mM crotonate for 6 days before analysis by microscopy. Untreated cells served as control. Scale bar, 100 μm. F RT-qPCR analysis of the indicated marker genes using cells from (E). OCT4 and NANOG, pluripotency markers. GATA6 and SOX17, endoderm markers. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. NS, not significant, **p < 0.01. G, H GAPDH KD cells stably expressing wildtype or K194R/K219R double mutant GAPDH were examined for GAPDH expression by RT-qPCR (G) and GAPDH activity (H). Error bars represent mean ± S.D. (n = 3 independent experiments). I RT-qPCR analysis of the expression of the indicated marker genes in control, KD cells, and GAPDH KD cells stably expressing wildtype or K194R/K219R double mutant GAPDH. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01. J Cells from G were cultured in regular endoderm differentiation medium for 2 days before expression analysis of the indicated marker genes. OCT4 and NANOG, pluripotency markers. GATA4, GATA6, and LHX1, endoderm markers. Error bars represent mean ± S.D. (n = 3 independent experiments). Significance was calculated using unpaired t test. *p < 0.05; **p < 0.01