Fig. 2.

SALL3 regulates differentiation into the three germ layers. a Venn diagrams illustrate overlaps among differentiation propensity marker candidate genes. SALL3 was the only gene showing an inverse correlation between ectoderm and mesoderm/endoderm differentiation. All candidate genes of differentiation propensity markers were listed in Supplementary Data 3. b Microarray data of SALL3 expression in ten hiPSC lines (n = 6, biological replicates). c SALL3 knockdown was confirmed by qRT-PCR analysis (n = 3, biological replicates). ^*P < 0.01, two-sided t test. d Western blot analysis of the total extracts obtained from control and SALL3 knockdown cells. LSD1 and β-actin were used as a nuclear protein control and loading control, respectively. Molecular weight is indicated as Mr (k). e qRT-PCR analysis of undifferentiated hPSC markers, OCT3/4 and NANOG. Total RNA was isolated from 253G1 SALL3 shRNA cells and 253G1 control shRNA cells in the undifferentiated state (n = 3, biological replicates). f–h qRT-PCR analysis of three germ layer-specific genes in EBs derived from 253G1 SALL3 shRNA cells and 253G1 control shRNA cells (n = 6, biological replicates). Ectoderm marker genes (f), endoderm marker genes (g), and mesoderm marker genes (h) are shown. ^*P < 0.05, ^**P < 0.01, two-sided t test. Error bars represent mean ± SD