Figure 3.
Pum1 Targets Naive Pluripotency Factor mRNAs during Exit from Self-Renewal
(A) siRNA against Pum1, but not Pum2, results in delayed downregulation of Rex1GFP expression 24 hr after 2i withdrawal.
(B) Pum1 knockdown results in a severe commitment defect. ESC colonies were stained with AP.
(C) Western blot confirming absence of protein in CRISPR Pum1 mutant ESCs.
(D) Impaired Rex1GFP downregulation in Pum1 mutant ESCs 48 hr after 2i withdrawal.
(E) RIP experiment showing Pum1 binding to predicted Pum1 target mRNAs and control transcripts without Pum1 binding site. Rabbit IgG was used as negative control. Error bars show standard deviation between technical triplicates. Replicate experiments yielded equivalent results.
(F) Gene expression profiles of indicated genes in a 48 hr N2B27 differentiation time course. Error bars represent standard deviation. Expression levels were normalized to Gapdh.
(G) Rex1GFP profiles for cells transfected with indicated siRNAs 24 hr after 2i withdrawal.
(H) Codepletion of Tbx3 restores differentiation in Pum1 siRNA transfected cells in a commitment assay.
(I) Tbx3fl/fl and Tbx3−/− ESCs were transfected with the indicated siRNAs and tested in a commitment assay. AP staining was performed after 4 days in 2i/LIF. Duplicate experiments are shown.
See also Figures S3 and S4.