VEGFR knockdown recapitulates the effect of sunitinib. (A, B) Validation of the shRNAs targeting VEGFRs by qRT-PCR and western blot. (C) Morphology of E14 cells expressing the indicated shRNA cultured in mES media without LIF. Cells treated with sunitinib or 2i were used as positive control. Scale bar, 50 μm. (D, E) qRT-PCR analysis of pluripotency genes (Oct4, Sox2, Nanog, Rex1) (D) and lineage-specific genes (mesoderm: Brachyury and SMA, endoderm: GATA4, ectoderm: Nestin) (E) in cells expressing the indicated shRNA cultured in mES media without LIF (passage 4). Cells treated with sunitinib or LIF were used as positive control. Data are mean SEM (n = 3). ##P < 0.01, ###P < 0.001 vs LIF (+) condition; *P < 0.05, **P < 0.01, ***P < 0.001 vs scramble shRNA. (F) E14 cells expressing the indicated shRNA were cultured in LIF-free mES media for 3 days at an initial density of 10 000 cells/well in 24-well plate. The percent of undifferentiated and differentiated colonies were calculated according to AP staining and morphology. Independent experiments were repeated at least three times. (G) Immunofluorescence staining of pluripotency markers (Oct4, Nanog, SSEA1) in cells described in E (passage 4). Cell nuclei were stained with Hoechst. Scale bar, 80 μm. (H) Western blot analysis of VEGFR1 and 2 in cells transfected with scramble shRNA, or shRNAs targeting VEGFR1 or 2 for 48 h. (I) Morphology and AP staining of E14 cells cultured in mES medium without LIF but containing 2i (3 μM PD0325901 and 3 μM CHIR99021), sunitinib (1 μM) or DC101 (20 μg/ml) for 3 days. (J) The percent of undifferentiated and differentiated colonies corresponding to I.