Fig. 5
FCCP-induced activation of CaMKII-MEK-ERK-Drp1 is impaired in KI MEFs
(A) Left: Representative immunoblots of WT and KI MEFs treated with FCCP for 0, 5, 15 and 30 min, showing levels of phosphorylated and total CaMKII, MEK, ERK and Drp1, with α-tubulin as loading controls. Right: Diagram showing cellular events occurring in WT MEFs with exposure to FCCP: acute cytosolic Ca2+ rise, followed by activation of CaMKII, MEK, ERK and Drp1. (B-E) Quantitation of phosphorylated CaMKII, MEK, ERK and Drp1 normalized by their respective total proteins in WT and KI MEFs following FCCP treatment (N = 4 experiments). (F) Percentage clearance of mitochondria over 24 h in WT and KI MEFs is expressed as percentage decrease in mito-PAmCherry fluorescence from its own baseline fluorescence intensity at 0 h by flow cytometry (N = 5 experiments, 10,000 cells per N). (G) Calculated mean slope from 0 to 24 h showing average percentage decrease in mito-PAmCherry fluorescence per hour. (H) Representative immunoblots of KI MEFs pre-treated with either LRRK2 kinase inhibitor, MLi-2 (30 nM), or vehicle, DMSO, for 1 h prior to FCCP treatment (10 µM) for 0, 15 and 30 min (N = 4 experiments). WT MEFs were pre-treated with DMSO as a positive control, followed by FCCP treatment of same duration. (I) LRRK2 kinase inhibition by MLi-2 was confirmed by the reduction in p-Rab10 (Thr73) level. Quantitation of phosphorylated (J) MEK and (K) ERK normalized by their respective total proteins following FCCP treatment. Data are presented as mean ± SEM. Statistical analyses: (B-E) Unpaired parametric Student’s t-test, each timepoint compared to its own respective baseline value at 0 min; (C) Unpaired parametric Student’s t-test between baseline values of WT and KI MEFs, #p < 0.05; (F, G) Unpaired parametric Student’s t-test; (I) One-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01 and ***p < 0.001. ns, not significant