Figure 4

Inhibition of apoptosis by TUDCA was not associated with a decrease in proapoptotic miRNAs expression. Mouse NS cells with 3 days of differentiation were either untreated or treated with 50 μM of TUDCA for 72 hours. Collected cells were stained with Annexin-V-APC/PI to evaluate cell death, or processed for total RNA extraction and miRNAs expression evaluation by quantitative Real Time-PCR. A) Representative Annexin V-APC/PI stainings showing decreased cell death after TUDCA incubation. B) Quantitation of either dying (Annexin+/PI-) or dead (Annexin+/PI+) cells depicted in FACS diagrams. Results are mean ± SEM of triplicates. C) Expression of proapoptotic miRNAs at 3 and 6 days, with or without TUDCA treatment. miR-16, let-7a and miR-34a 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 differentiated cells at 3 days as calibrator. Data represent mean ± SEM of three independent experiments. *p < 0.05 compared to respective nontreated cells at 6 days.