Fig. 2.

USP26 is required for ESC differentiation. a Real-time qPCR analysis of mouse Usp26 mRNA expression in MEFs, iPSCs, and ESCs, **p < 0.01 compared to MEFs. b Western blot analysis of USP26 protein expression in MEFs, iPSCs, and ESCs. c Real-time qPCR analysis of mouse Usp26 and Nanog mRNA expression during Dox-induced OSKM-mediated MEF reprogramming on days 0, 4, 8, and 12, *p < 0.05 compared to Usp26, **p < 0.05 compared to day 0. d Real-time qPCR analysis of mouse Usp26 and Nanog mRNA expression in ESCs after LIF withdrawal and treatment with 1 μM RA on days 1, 2, 3, and 4, *p < 0.05 compared to Usp26, **p < 0.05 compared to day 1. e Western blot analysis of USP26, OCT4, and NANOG protein expression in ESCs after LIF withdrawal and treatment with 1 μM RA at days 0, 2, 4, and 6. β-actin was used as a loading control. f Experimental scheme of Usp26-induced ESC differentiation. ES cells were transduced with Dox-inducible GFP-tagged Usp26 or empty vector lentivirus. After ESC colonies formed, then low-dose Dox (0.1 µg/ml) was added for 1 day pre-selection. Day 0 was defined as the day when pre-selected GFP-positive colonies were cultured in iSF1 medium with high-dose Dox (2 µg/ml), individual colonies were tracked and taken pictures on Usp26-expressing days 1, 2, 3, and 4. g Bright field (BF) and fluorescent microscopic images of ESC morphology with Dox-inducible GFP-tagged mUsp26 overexpression or with GFP-tagged empty vector (EV). h Bright field images of ESC morphology of wild-type (WT) or Usp26 knockout (KO) ESCs treated with RA. WT or Usp26 KO ESCs were cultured in ES differentiation medium (LIF withdrawal with 1 μM RA). Individual colonies were tracked and photographed on days 0, 2, 4, and 6 under microscope. The data are presented as means ± SD from three independent experiments. a, c, d Two-way ANOVA for multiple comparisons