Fig. 4

Characterization of chemically induced differentiation by qRT-PCR and immunoblotting. a D12 cultures were collected and analyzed by qRT-PCR for markers of cell identity genes, transcription factors, and signaling pathways. Data is presented as mean ± SEM (n = 4, independent experiments). *P ≤ 0.05 (Wilcoxon test vs DMSO control). b Modulation of GATA4 isoforms by compound treatment in differentiating mESCs. Differentiating mESCs were treated with 3i-1000 during D2–D10 window of differentiation and collected for immunoblotting at D5 and D12. Anti-GATA4 detection, lanes 1–4 (day 5 samples); lane 1: medium only (M), lane 2: DMSO (D), lane 3: 3i-1000 (3 μM), lane 4: 3i-1000 (5 μM), and lanes 5–8 (D12 samples) lane 5: medium only (M), lane 6: DMSO (D), lane 7: 3i-1000 (3 μM), lane 8: 3i-1000 (5 μM). Note the heavier GATA4-70 kDa band is abundant in differentiating mESCs. c For confirmation of this band as GATA4, overexpression of GATA4-V5 in HEK293 cells and immunoblotting for anti-GATA4 and anti-V5. Lane 1: protein ladder, lane 2: GATA4-V5, lane 3: GATA4-V5 (1/5), and lane 4: GATA4-V5 (1/25). Note that GATA4-70 kDa is detected in cells with overexpression by both anti-GATA4 and anti-V5 antibodies