Supplementary Figure 4: SDH drives an inflammatory phenotype in PPBL B cells
From: SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1–Nrf2
(a) Production of IL-6 by MZ-like B-LCLs from HCs (n = 3), quantified in cell culture supernatants of cells activated for 48 hours with IL-21 plus CD40L, in presence or absence of monomethyl fumarate. (b) Production of IL-6 by Patients B-LCLs (n = 3), quantified in cell culture supernatants of cells cultured for 24 hours in presence or absence of UK-5099 (1, 10 μM), BPTES (1, 10 μM) or dichloroacetate (DCA; 0.1, 0.5 mM). (c) OCR and ECAR measurements of primary B cells from healthy controls (n=16) and primary B cells from Patient #15 (PPBL patient). Basal values are shown. (d) Number of hydrogen bonds formed over time between SDHB and residues 43–46 of SDHA, wild type (wt vs. A45T). Red segments highlight the trajectory extract shown in Supplementary Video V1. Corresponding frequency distributions of the counted hydrogen bonds are shown in the insets. Experiment was repeated independently 12 times with similar results. (e) Frequency distributions of the residues from the N-terminal SDHA (left) and SDHB (right) involved in inter-domain hydrogen bonds in simulations with SDHA wt and SDHA-A45T. (f) Time fraction of simulated contacts between Ala/Thr45 and adjacent SDHA residues (Arg458, Phe459, Asp511, Arg512 and Met514), depicted as a percentage of the SDHA–SDHB interaction time in wt and A45T. Experiment was repeated independently 12 times with similar results. (g) Visualization of Sanger sequencing of B-LCL derived cDNA of Patient 8, carrying the A45T mutation (left). Asterisk indicates changes of nucleotide G to A at position 133. Compound SDHA plus SDHB activity (enzyme complex derived from B-LCLs) from Patient 8 relative to HCs (n = 3) (right). Compound SDHA–SDHB activity was independently tested 4 times in Patient 8 and in the control subject. Pooled data are represented as mean ± SEM. Statistical significance assessed by two-sided paired t-test (a), two-sided unpaired t-test (g), one-way ANOVA, adjustments were made for multiple comparisons with BH procedure (f). * p < 0.05, ** p < 0.01, ns, not significant.
