(A) qRT-PCR on Mir182 and Mir183. For each miRNA, the expression level in wild-type (WT) cells was set as one for relative comparison. U6 RNA was used for normalization. The results represent the means (± SD) of three independent replicates. (B) Western blotting in the WT, Mir182Δ, Mir183Δ, and Mir182Δ/Mir183Δ mESCs. GAPDH was used for normalization in calculating the relative expression levels. (C) Colony formation assay for mESCs. The mESCs were cultured in 15% FBS+ Lif for 7 days, and the resultant colonies were fixed and stained for alkaline phosphatase (AP). (D) Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i + Lif medium for 5 days. The resultant colonies were fixed and stained for AP. In (C and D), the colony morphology and AP intensity were evaluated through microscopy; 100–200 colonies were examined each time to determine the percentage of undifferentiated colonies. The results represent the means (± SD) of three independent experiments. (E) Western blotting of pluripotency factors during embryoid body (EB) formation.