Fig. 2.

Cops5 is required for ESC self-renewal and differentiation. (A) iC5; C5 KO ESCs cultured with Dox (Passage 0, P0) were switched into medium without Dox for two passages. The protein levels of the pluripotency factors Nanog and Oct4, as well as the CSN subunits, Cops2, Cops5, and Cops8, were measured by Western blot. (B) Colony morphology change upon Cops5 KO. Phase-contrast images of ESC colonies at each passage are shown. (Scale bar, 100 μm.) (C) Growth curves of Cops5 KO ESCs. iC5; C5 KO ESCs were cultured in ESC medium with or without Dox. The cell numbers were counted every passage, and an equal amount of ESCs was plated into tissue culture dishes. (D) Colony forming assay of iC5; C5 KO ESCs with or without Dox. (Scale bar, 200 μm.) (E) Quantitative RT-PCR was performed to measure the RNA levels of the pluripotency markers Nanog, Oct4, and Sox2. (F) Expression of differentiation genes after Cops5 KO. (G) iC5; C5 KO ESCs were cultured with Dox, and then the cells were used for EB differentiation with or without Dox. (Left) The images of day 4 EBs (40× magnification) with or without Dox. The relative diameters of day 4 EBs were measured and plotted (Right). (H) Quantitative RT-PCR analysis of pluripotency and differentiation genes in day 4 EBs, as described in G. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.