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Fig. 4 miR-1 affects the apoptosis of neural crest cells via the mitochondrial apoptosis pathway and caspase-3. (A) Detection of apoptosis on day 3 following miR-1 inhibitor transfection using AO/EB staining. Scale bar, 100 µm. (B) Quantitative analysis of AO/EB staining. (C) TUNEL staining of NCCs. Scale bar, 100 µm. (D) Quantitative analysis of TUNEL staining. (E) Protein levels of the mitochondrial apoptosis pathway markers BCL-2, pro- and cleaved-caspase-3, pro- and cleaved-caspase-9 and PARP detected via western blotting. (F-K) Quantification of the protein levels of (F) BCL-2, (G) pro-caspase-9, (H) cleaved-caspase-9, (I) pro-caspase-3, (J) cleaved-caspase-3 and (K) PARP, which are presented in (E). (L) miR-1 expression assessed by reverse transcription-quantitative PCR. (M) Expression levels of pro- and cleaved-caspase-3 after transfection with the miR-1 mimic. (N and O) Quantification of the protein levels. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. Control. miR-1, microRNA-1; PARP, poly ADP-ribose polymerase, AO, acridine orange; EB, ethidium bromide.