Figure 5.

Ythdf1, Ythdf2, and Ythdf3 are functionally redundant in ESC differentiation. (A) Teratomas generated from KO cell line and from WT control. Single-KO teratomas show all germ layers, while triple-KO (TKO) teratomas are poorly differentiated. Selected differentiated structures are marked by arrows. (B) Immunostaining of triple-KO and WT control to Oct4 (red), Foxa2 (green), Tuj1(purple) and DAPI (blue). Triple-KO teratomas contain patches of Oct4 staining, unlike WT teratomas. (C) Alkaline phosphatase (AP) staining of disassociated teratomas from triple-KO and WT control samples, showing a greater AP staining in the Ythdf triple-KO. (D) RT-PCR of pluripotency genes (left) and differentiation genes (right), measured in WT mESCs, and mEBs from the following cell lines: WT control, Ythdf single-KO, Ythdf triple-KO, and Ythdf triple-KO + overexpression (OE) of Ythdf1/2/3 (rescued cell lines). Triple-KO EBs express pluripotent markers and repress differentiation markers similarly to mESCs. Rescued EBs express differentiation markers, similarly to single-KO EBs. (E) EBs were induced for 7 d from the indicated cell lines, and dissociated into single cells at day 7 and comparable amounts of cells were replated in ES medium. Undifferentiated AP⁺ colonies number per 1000 plated cells was evaluated 5 d later.