MCU suppression inhibits the mitochondrial calcium spike without inhibiting the actin burst. U2OS cells were transfected with either scrambled siRNA (control) or siRNA against MCU (MCU KD). After 48 h, cells were transfected with mito-R-GECO (mitochondrial calcium) and GFP-Ftractin (polymerized actin) or cyto-R-GECO (cytoplasmic calcium) and imaged at 72 h after siRNA treatment. (A) Confocal image montages of cells at indicated times after 4 µM ionomycin stimulation. For MCU KD, the mitochondrial calcium signal is enhanced to reveal the faint mitochondrial outline. Arrowheads show mitochondrial calcium rise, and arrows denote polymerized actin increases. Bars, 5 µm. (B) Quantification of the mitochondrial and cytosolic calcium spikes upon stimulation with 4 µM ionomycin (at time 0); n = 10 cells (Mito calcium control), 18 cells (Mito calcium MCU KD), 10 cells (Cyto calcium control), or 15 cells (Cyto calcium MCU KD). Error bars represent SEM. (C) Quantification of the actin burst upon stimulation with 4 µM ionomycin (at time 0); n = 10 cells (control) or 15 cells (MCU KD). Error bars represent SEM.