Extended Data Fig. 6: Mitochondrial matrix acidification promotes mitochondrial Na+/Ca2+ exchange via the NCLX.
From: Na+ controls hypoxic signalling by the mitochondrial respiratory chain
a, TEM-EDX determination of calcium element (Ca) versus carbon (C), oxygen (O) and lead (Pb) content in regions of mitochondria with (empty circles) or without (filled circles) electron dense spots (n = 7). b, Frequency of CaP precipitates per mitochondrion in BAECs, seen by TEM during normoxia (741 mitochondria), 10 min of hypoxia (1% O2; 619 mitochondria) or 30 min with 1 μM FCCP (393 mitochondria), three independent experiments. c, Total Ca2+ content of mitochondria extracted from mouse adult fibroblasts (MAFs) which had been treated for 10 min with 1 μM FCCP or 1 μM rotenone, measured in a hypotonic buffer at pH 6.8 (n = 4). d, Western blot showing MCU and Fp70 in CRISPR Control, MCU KO2 and MCU KO3 cells (representative image of two independent experiments). e, Mitochondrial Ca2+ measured by live cell confocal microscopy in MCU WT or KO human breast cancer cells transfected with Cepia2mt (fluorescence signal relative to starting signal –F/F0–, representative traces of n = 5 for WT, n = 9 for MCU KO2 and n = 5 for KO3, independent experiments). f, Total Ca2+ content of mitochondria extracted from MAFs which had been subjected to normoxia or 10, 30 or 60 min of hypoxia (1% O2), measured in a hypotonic buffer at pH 6.8 (n = 5). g, Ca2+ content of mitochondria extracted from MAFs which had been subjected to 10 min normoxia or 10 min hypoxia (1% O2) in a medium without Ca2+, measured in a hypotonic buffer at pH 6.8 (total mitochondrial Ca2+) or pH 7.5 (soluble mitochondrial Ca2+; n = 4). h, Cytosolic Ca2+ measured by live cell confocal microscopy in MEFs transfected with cyto-GEM-GECO in the absence of Ca2+ in the incubation medium (n = 4). i, Endoplasmic reticulum Ca2+ measured by live cell confocal microscopy in MEFs transfected with erGAP3 in the presence or absence of Ca2+ in the incubation medium (n = 8). j, Superoxide detection with DHE in MCU WT or MCU KO immortalized breast cancer cells in normoxia (Nx) or after 10 min of hypoxia (1% O2; Hp; n = 4). All data except e are represented as mean ± s.e.m. One-way ANOVA with Tukey’s test (c and f) and two-tailed Student’s t-test (a, b, g and j): n.s. not significant, * P < 0.05, ** P < 0.01. Two- tailed Student’s t-test (CRISPR Control Hp vs MCU KOs Hp in j): & P < 0.05, && P < 0.01. For gel source data, see Supplementary Fig. 1.
