Fig. 1

Identification of USP26 as a negative regulator of pluripotency. a Schematic of experimental strategy for screening essential Usps for the generation of iPSCs. Dox-inducible OSKM-transgenic MEFs were plated, transfected with 48 individual mouse Usp shRNA lentiviral vectors to knockdown Usps, and stained for AP⁺ iPSC colonies after 12 days of Dox treatment. b Quantification of AP⁺ colonies after 12 days of OSKM induction in MEFs transduced with shRNA, targeting members of the mouse Usp family, p < 0.001 compared to control shRNA. c Quantification of AP⁺ colonies after 12 days of OSKM induction in MEFs transduced with mouse Usp26 shRNA, Usp20 shRNA, or control shRNA lentivirus, *p < 0.05 compared to control shRNA, **p < 0.01 compared to control shRNA. d Quantification of AP⁺ colonies after 12 days of OSKM induction in MEFs transduced with pLtet-O (tetracycline-inducible) mouse Usp26, Usp20, or empty vector (EV) lentivirus, **p < 0.01 compared to EV. e Images of AP staining of iPSC colonies after 12 days’ OSKM induction in MEFs transduced with control shRNA, Usp26 shRNA, EV, or pLtet-O lentivirus overexpressing Usp26. f Bright field (BF) and immunofluorescence microscopic images of Oct4, Nanog, and Dppa4 in Usp26 knockdown iPSCs. iPSC colonies were fixed, blocked, and stained with specific antibodies, followed by goat anti-mouse antibody-conjugated Texas Red. Nuclei were stained with DAPI. Scale bar, 100 µM. The data are presented as means ± SD from three independent experiments. b–d Two-way ANOVA for multiple comparisons