Overexpression of miR-96 family members represses neural specification. (A) hESCs with overexpression of miR-96 (indicated by GFP) exhibited a flat morphology, as shown by phase-contrast microscopy, and retained the expression of POU5F1 (red) after 10 days of neural differentiation. Scale bars: 50 μm. (B) The protein levels of PAX6 and POU5F1 were analyzed by western blotting at day 10 of differentiation of miR-96- or miR-182-overexpressing hESC lines. The GFP-hESC line (without miRNA insertion) was used as a control. (C) The fold changes in expression of neuroectodermal markers (PAX6, SOX1, CDH2) and pluripotent cell markers (POU5F1 and NANOG) were quantified by qPCR during differentiation of miR-96-overexpressing and GFP control hESCs. The relative gene expression levels were normalized to day 6 GFP control cells. qPCR data are presented as mean±s.e.m. from triplicate samples (n=3, *P<0.05).