Figure 3 Loss of MICU1 leads to a deficit in mitochondrial calcium handling without impairing respiration Loss of MICU1 leads to a deficit in mitochondrial.

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Figure 3 Loss of MICU1 leads to a deficit in mitochondrial calcium handling without impairing respiration Loss of MICU1 leads to a deficit in mitochondrial calcium handling without impairing respiration (A) Oxygen consumption rates (OCR) and (B) extracellular acidification rates (ECAR) of MICU1+/− and MICU1−/− fibroblasts were measured by Seahorse XF analysis. Mean values ± SE (error bars) from 3 independent experiments are shown (differences are not statistically significant). (C) Immunoblot of whole cell lysates from MICU1+/− and MICU1−/− fibroblasts. (D) Digitonin-permeabilized fibroblasts were given a pulse of 35 μM CaCl2 while monitoring extramitochondrial Ca2+ with calcium green-5N.8 (E) Quantification of the rate of calcium uptake from calcium uptake traces including those in D (using linear fit from 30 to 40 seconds). Mean values ± SD (error bars) from 3 independent experiments are shown. (F) MICU1+/− (+) and MICU1−/− (−) fibroblasts were exposed to different conditions (untreated, 500 nM CCCP, 1 μM oligomycin, or serum starvation) and pyruvate dehydrogenase (PDH) and phosphorylated PDH (at S293) were measured by immunoblot after lysing the cells in 1% Triton-X-100, 150 mM NaCl, 25 mM Hepes pH 7.4, and protease and phosphatase inhibitors. (G) Quantification of 3 independent experiments including the representative experiment in F are shown. Mean values ± SE (error bars). MCU = mitochondrial Ca2+ uniporter; SDHB = succinate dehydrogenase subunit B. David Lewis-Smith et al. Neurol Genet 2016;2:e59 © 2016 American Academy of Neurology