Fig. 9: PrPC recruits miR-214-3p into its phase-separated condensate in living skeletal muscle cells, which in turn promotes pathological aggregation of PrP.
a Western blot for PrP in the sarkosyl-insoluble pellets and the corresponding cell lysates from C2C12 cells stably overexpressing mCherry-Cry2-WT PrPC transfected without (-) or with (+) 10 μM miR-214-3p and cultured for 12 h after activation by 488-nm laser for 10 min. β-actin served as the protein loading control. b The normalized amount of insoluble PrP aggregates 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. c The cell viability (%) (solid black circles shown in scatter plots) was measured by MTT reduction assay and expressed as the mean ± SD (with error bars) of values obtained in five independent experiments. C2C12 cells expressing mCherry-Cry2 (gray) or expressing mCherry-Cry2-WT PrPC transfected without (-) (orange) or with (+) (blue) 10 μM miR-214-3p (b, c). Immunogold electron microscopy of PrP fibrils purified from the same C2C12 myoblasts stably expressing mCherry-Cry2-WT PrPC transfected without (d) or with (e) 10 μM miR-214-3p as in (a), and labeled by gold particles conjugated with anti-PrP antibody. Scale bars, 100 nm. f Western blot for PrP in the sarkosyl-insoluble pellets and the corresponding cell lysates from C2C12 myoblasts stably overexpressing WT PrPC incubated with differentiation medium for 2 days, transfected without or with 10 or 20 μM miR-214-3p and cultured for 48 h. β-actin served as the protein loading control. g the same as in (b). C2C12 cells expressing WT PrPC transfected without or with 10 (gray) or 20 μM (orange) miR-214-3p. b, c, g 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. h Western blot for PrP in the cell lysates from the same C2C12 cells stably overexpressing mCherry-Cry2-WT PrPC transfected without or with 10 μM miR-214-3p as in (a), and digested with 0.25, 0.50, 0.75, 1.0, and 2.0 μg/ml proteinase K.
