Fig. 3

Combination of CQ/IH induces phosphorylation and mitochondrial translocation of CaMKII (Thr286) and Drp1 (Ser616). MDA-MB-231 cells were treated with CQ (20 μM) in the presence or absence of IH (10 μM) for 48 h. a Mitochondrial morphology was observed by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars: 10 μm. b Mitochondrial length was measured with ImageJ software. 50 cells from three independent experiments (mean ± SD, ^***P < 0.001 compared with control). c Whole cellular lysates (WCL) and cytosolic (Cyto)/mitochondrial (Mito) fractions were prepared and subjected to Western blot using antibodies against phospho-Drp1 (p-Drp1) (S616), p-Drp1 (S637), and Drp1. d The colocalization of Drp1 (green) and MitoTracker (red) was examined using confocal microscopy. Scale bars: 10 μm. e The expression of p-CaMKII and CaMKII in WCL, Cyto, or Mito was examined by Western blot. f The colocalization of CaMKII (green) and MitoTracker (red) was examined by confocal microscopy. Scale bars: 10 μm. g and h The calcium ion level was analyzed by flow cytometry. The values represent the mean ± SD for three separate experiments (^***P < 0.001 compared with control). i Whole cell lysates were prepared and subjected to immunoprecipitation using anti-CaMKII; the associated CaMKII and Drp1 were determined using immunoblotting. j The colocalization of CaMKII (red), Drp1 (green), and MitoTracker (blue) was examined using confocal microscopy. Scale bars: 10 μm