Supplementary Figure 3: IRE1α modulates mitochondrial bioenergetics under ER stress.

(A) ATP measurements in cells treated with 100 ng/ml tunicamycin (Tm) for the indicated times (NT: n = 18; 4h: n = 12; 24h: n = 8; all n are biologically independent experiments). (B) IRE1α KO cells reconstituted with IRE1α-HA or Mock or CRISPR cells (C) were processed for western blot analyses to monitor levels of indicated proteins. Cells were treated with 100 ng/ml Tm for the indicated times (n = 3 independent experiments). (D) IRE1α KO cells were reconstituted with IRE1α-HA and processed to obtain purified MAM fractions after 4 h treatment with 1 µg/ml Tm. Fractions were analysed by western blot (WB) for indicated UPR and MAM markers (Cr: crude mitochondria; H: homogenate; M: MAMs; P: pure mitochondria; C: cytosol; E: ER) (n = 2 independent experiments). (E) Same cells described in D were treated with 0.1 µg/ml Tm for indicated time points and stained for ERp72, TOM20 and HA using indirect immunofluorescence. Scale bar = 20 µm. Confocal microscopy analysis and co-localization coefficient was calculated (n = 3) (total cells analysed for t = 0 and 16; n = 40; t = 8; n = 43). (F) IRE1α KO cells reconstituted with IRE1α-HA or empty vector (Mock) were processed to obtain subcellular fractions and analysed by WB to monitor the levels of indicated proteins. Right panel: quantification in the expression of IP₃R3 normalized to calnexin (CNX) in the ER fraction (n = 6 independent experiments). (G) IRE1α KO cells reconstituted with IRE1α-HA or empty vector (Mock) transiently expressing control or 9x MAMs linker were imaged for TEM to measure MAMs width. Graph represents the frequency distribution of MAMs width. All plots represent mean and SEM. Statistical differences were detected with unpaired Student´s t-test; two-way ANOVA (A). Source data have been provided in Supplementary Table 6.