Drp1-S600 phosphorylation is required for the effects of high K⁺ on mitochondrial dynamics. (A) HeLa cells were first transfected with control siRNA (siCont; Invitrogen) and siRNA for Drp1 (siDrp1) before transfection with the Drp1 plasmids. 1 d later, HeLa cells were transfected with GFP, YFP-Drp1^R, YFP-Drp1^R-S600A, or YFP-Drp1^R-S600D together with pDsRed2-Mito. Proteins were separated by SDS-PAGE and analyzed by immunoblotting using Drp1 and actin antibodies. (B) HeLa cells were treated without (No sti) or with 20 mM K⁺ (high K⁺), and mitochondrial fluorescence was analyzed by time-lapse imaging. Mitochondrial length was measured through analysis of the elongation index. Bars, 5 μm. (C) The elongation index was normalized to the value before stimulation. Values shown are the mean ± SEM (n = 5) in each group. Values were analyzed using one-way ANOVA followed by post-Tukey test. *, P < 0.01. (D) Neurons (10 DIV) were cotransfected with pDsRed2-Mito and GFP, YFP-Drp1, or YFP-Drp1-S600A as indicated. Neurons (at 11 DIV) were then treated without (No sti) or with 45 mM K⁺ for 15 min, and mitochondrial fluorescence was analyzed by time-lapse imaging. Bars, 10 μm. (E) Mitochondrial length was measured through analysis of the elongation index. Values shown are the mean ± SEM (n = 5) in each group. Values were analyzed using one-way ANOVA followed by post-Tukey test. *, P < 0.05; **, P < 0.005.