Figure 4.

Effects of Zic3/Tfe3 Heterozygous Deletion on Pluripotency Exit

(A) Western blot analysis for ZIC3 in iPSCs and ESCs grown in S/L. Right: quantification using actin as loading control. Three XY versus 3 XX iPSC lines, and 1 XY versus 1 XX ESC line. ^∗∗p < 0.01, unpaired two-tailed t test. Data from a representative experiment from at least three independent experiments.

(B) Scheme of heterozygous Zic3 deletion strategy in XX ESCs followed by LIF withdrawal.

(C) Western blot analysis for ZIC3 in two independent Zic3^+/− ESC lines, Zic3^+/+ ESCs and XY ESCs all grown in S/L (n = 1). KO, knockout; WT, wild-type.

(D) qPCR analysis for X-chromosome DNA copy number. X copy numbers are presented as the average ratio of genomic DNA (gDNA) quantities for four X-linked genes (Tfe3, Bcor, Pdha1, and Mid1) to gDNA quantities for autosomal gene Gapdh (n = 2).

(E) qRT-PCR analysis for pluripotency-associated gene expression during LIF withdrawal in the two independent Zic3^+/− XX ESC lines, the Zic3^+/+ XX parental ESC line, and an XY ESC line (n = 2).

(F) Scheme of heterozygous Tfe3 deletion strategy in XX ESCs followed by LIF withdrawal.

(G) qRT-PCR analysis for Tfe3 expression in the Tfe3^+/− XX ESC line, the Tfe3^+/+ XX parental ESC line, and an XY ESC line (n = 1).

(H) As in (E) for Tfe3^+/− ESCs.

Results are presented as averages (±SEM) of biological duplicates (n = 1). See also Figures S3 and S4.