Extended Data Fig. 5: Acute hypoxia promotes A/D transition in CI and matrix acidification independently of NCLX activity.
From: Na+ controls hypoxic signalling by the mitochondrial respiratory chain
a–c, ND3·Cys39 exposure, which reflects the D conformation of CI, measured as the ratio between TMR signal (Cys39 labelling) and Sypro Ruby staining (total protein for the ND3 band, identified by mass spectrometry11). Thermal deactivation is used as a positive control of CI D state. Scheme of the technique11 (a); BAECs (b; n = 5 and n = 4 for de-activated samples) or HepG2 (c; n = 2) exposed to normoxia (Nx), 5 min of hypoxia (1% O2, H5), normoxia with CGP-37157 (NxCGP), 5 min of hypoxia with CGP-37157 (H5CGP). d, Complex I reactivation rate measured in the presence of Mg2+ in isolated mitochondrial membranes from BAECs subjected to normoxia, 10 min of hypoxia (1% O2) and NCLX inhibition with CGP-37157 (n = 4). e–g, Mitochondrial matrix pH measured using calibrated mitosypHer in WT or CI KO cybrid cell lines (e; n = 8), MEFs preincubated or not with rotenone (f; n = 4) or NCLX WT or KO MEFs (g; n = 8). h, Mitochondrial matrix acidification using mitosypHer in BAECs in normoxia or acute hypoxia (1% O2) treated or not with NCLX inhibitor CGP-37157; time-course traces and slopes (n = 4). i, j, Effect of the CI inhibitor rotenone on cytosolic Ca2+ (i) or cytosolic Na+ (j) measured by live confocal microscopy with Fluo-4 AM or CoroNa Green AM, respectively, in BAECs in normoxia or acute hypoxia (1% O2), n = 3. All data are represented as mean ± s.e.m. Two-tailed Student’s t-test (b, d, h, i, j): n.s. not significant, * P < 0.05, **P < 0.01, *** P < 0.001.
