miR-16, let-7a and miR-34a are increased during mouse ES cell differentiation. Mouse ES cells (Sox1-GFP 46C) were differentiated using an adherent monolayer protocol. Cells at 4 and 8 days were collected for total RNA extraction and subsequently processed for evaluation of specific differentiation markers, as well as proapoptotic miRNA expression by quantitative Real Time-PCR. A positive control for neural differentiation was also performed at day 8 by treating cells with either 10 nM LY411575 or 0.01%DMSO (control) for 12 hours. A) Semi-quantitative RT-PCR analysis for selected markers of lineage commitment in day 4 and 8, as well as in LY411575-treated and untreated cells. B) Representative bright-field, phase contrast images showing increased neuronal differentiation after LY411575 incubation compared with control (DMSO-treated) cells. C) Expression of miR-16, Let-7a and miR-34a at 4 and 8 days of ES cell differentiation and in control (DMSO-treated) and LY411575-treated rosette cultures at 8 days. miRNAs expression were evaluated from 10 ng of total RNA, using specific primers for each miRNA, and GAPDH for normalization. Expression levels were calculated by the ΔΔC t method using either differentiated cells at 4 days or LY411575-untreated cells as calibrator. Data represent mean ± SEM of three independent experiments. *p < 0.05 compared to respective nontreated cells. Scale bar: 50 μm. d, days.