miR-96 directly targets the PAX6 gene. (A) The nucleotide sequences of miR-96 family members. The seed sequence is marked in red. (B) Fold changes in miR-96 and miR-182 expression during NEP and EPI differentiation from hESCs, quantified by qPCR. (C) The miR-96 targeting sites TS1 and TS2 are located in PAX6 3′UTR. (D) The luciferase reporter assays were performed on TS1 and TS2 sites, and the whole PAX6 UTR. The complementary sequences of TS1 or TS2 sites with miR-182 are shown. (E) The luciferase reporter analyses were performed on mutant TS1, mutant TS2 sites or mutant PAX6 UTR (both TS1 and TS2 mutated). The mutant nucleotides are shown in red. (F) The luciferase reporter analyses were performed on wild-type and mutant PAX6 UTR reporter in EPI cells, which endogenously express miR-96 and miR-200 family members. The reporter without the UTR is used as a negative control, and the reporter with ZEB2 UTR as a positive control. The results are presented as mean±s.e.m. of three experiments (n=3, *P<0.05, Student's t-test).