Fig. 6: PrPC increases the stability of mature miR-214-3p to inhibit autophagy and myoblast differentiation. | Nature Communications

Fig. 6: PrPC increases the stability of mature miR-214-3p to inhibit autophagy and myoblast differentiation.

From: Excess PrPC inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

Fig. 6

Dual luciferase reporter assays, in which miR-214-3p (a) or miR-204-5p (b) represses the wild-type 3’ end of untranslated region (WT 3’UTR) of the gene ATG5 or LC3B associated with luciferase. The relative luciferase activity of the reporters after transfection with NC (gray), miR-214-3p mimic (a, orange), or miR-204-5p mimic (b, blue) at day 4 of differentiation in C2C12 myoblasts stably overexpressing WT PrPC or KO for PrPC (solid black circles shown in scatter plots), and the relative stability of miR-214-3p (c), miR-204-5p (d), and nonspecific miR-183-5p (e) in C2C12 myoblasts stably overexpressing WT PrPC (red) or KO for PrPC (blue) incubated with differentiation medium for 4 days and then treated with 20 mg/ml α-amanitin, were expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. p = 0.001, 0.268, 0.000226, 0.467 (a), 0.035, 0.926, 0.001, 0.372 (b), 0.048 (c), 0.000086 (d), and 0.742 (e), respectively. Western blot for ATG5 (f), LC3B (g), and MyHC after transfection with NC, miR-214-3p inhibitor (f), or miR-204-5p inhibitor (g) at 10 μM in the aforementioned cells upon differentiation for 4 days. β-actin served as the protein loading control.The relative amount of ATG5 (h), LC3B-II (j), or MyHC (i, k) in the aforementioned cells (solid black circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Transfection of NC, miR-214-3p inhibitor (h, i), or miR-204-5p inhibitor (j, k) in C2C12 myoblasts stably overexpressing WT PrPC (orange or blue) or KO for PrPC (gray). a–e Statistical analyses were performed using two-sided Student’s t-test. Values of p < 0.05 indicate statistically significant differences. The following notation is used throughout: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001 relative to controls. n.s., no significance. h–k One-way two-sided ANOVA and multiple comparisons with no adjustments were performed by SPSS 19.0, and different letters indicate significant differences at the level of p < 0.05.

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