Extended Data Fig. 6: Ether lipid synthesis is required for respirasome assembly from DHODH inhibition.
From: Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly
a, Western blot of FAR1 from cells treated with Brequinar in the presence or absence of uridine (n = 2 experiments). b, Western blot of FAR1 from sgFAR1 cells (n = 2 experiments). c, Lipidomic analysis of PC and PE subclass abundances in mitochondrial fractions isolated from sgFAR1 cells treated with Brequinar (min-max box plot, line at mean, n = 3 biologically independent samples, Student’s t-test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, * left to right sgControl DMSO [compared to sgControl Breq] q = 0.0003, 0.0034, 0.0007, sgControl DMSO [compared to sgFAR1 DMSO] q = 0.0208, <0.0001, 0.0005, 0.0016, 0.0016, 0.0001, sgControl Breq [compared to sgFAR1 Breq] q = <0.0001, <0.0001, <0.0001, <0.0001, <0.0001). d, Western blot of FAR1 from sgFAR1 cells exogenously expressing CRISPR-resistant FAR1 (n = 2 experiments). e, BN-PAGE and f, quantification of respirasome abundance (MTCO1/ATP5A intensity) of mitochondria isolated from sgFAR1 + exogenous FAR1 cells treated with Brequinar (n = 3 independent experiments, two-sided paired Student’s t-test, * left to right p = 0.0460, 0.0142). g, Western blot of AGPS from sgAGPS cells (n = 2 experiment). h, BN-PAGE and i, quantification of respirasome abundance of mitochondria isolated from sgAGPS cells treated with Brequinar (n = 4 independent experiments, two-sided paired Student’s t-test, * left to right p = 0.0445, 0.0005). j-l, Heatmaps of plasmenyl PC and PE (that is plasmalogens) and cardiolipin species in sgFAR1 mitochondria (n = 4 biologically independent samples). In this figure, cells were treated with Brequinar for 48 hours, heatmap row/column groupings are Pearson’s correlations, data are presented as mean values +/- s.e.m.
