Fig. 4

Heterozygous mutations of Klhl13 and Dusp9 in female mESCs partially phenocopy the male pluripotency state. a Schematic depiction of the strategies used to generate Klhl13 (K13) and Dusp9 (D9) mutant cell lines. b–f Comparison of female 1.8XX mESCs with a heterozygous (HET) or homozygous (HOM) deletion of Dusp9 (yellow), Klhl13 (blue) or both (red) with the parental XX line and XO controls (2 clones per genotype). Individual measurements are shown as gray dots (clone 1) and triangles (clone 2), and the mean across two clones and three biological replicates is indicated by a thick bar. b Immunoblot quantification of Dusp9 (top) and Klhl13 (bottom) protein levels, normalized to Tubulin and to the mean of the XX controls. c Quantification of MAPK target genes by qPCR. d Boxplots showing expression of Mek (left), Gsk3 (middle), and Akt (right) target genes in cell lines with the indicated genotypes as assessed by RNA-seq. Boxes indicate the 25th to 75th percentiles, and the central line represents the median. e Quantification of pMek, normalized to total Mek and to the XX control cells by immunoblotting. f Pluripotency factor expression (Nanog and Prdm14) assessed by qPCR. g qPCR quantification of pluripotency factors during differentiation by 2i/LIF withdrawal in one clone for each genotype from the cell lines used in b–f. Mean and SD of three biological replicates is shown. *p < 0.05 Wilcoxon rank sum test (d), otherwise two-tailed paired Student’s t test comparing each mutant/XO cell line and XX wildtype controls