Extended Data Fig. 1: Characterization of an inducible mouse model of Fh1 loss.
From: Fumarate induces vesicular release of mtDNA to drive innate immunity
a, Genomic DNA PCR using primers4 flanking exons 3 and 4 of Fh1 showing amplification product for the Fh1 wild-type allele (bottom band; Fh1+/+), the floxed (unrecombined) allele (top band; Fh1fl/fl) and the recombined allele (middle band; excised LoxP fragments; Fh1−/−) from kidney samples at day 5 post-induction. Mice carrying one allele of each Fh1+/+ and Fh1fl/fl i.e. Fh1+/fl; Rosa26-Cre-ERT2 were used for comparison with Fh1+/+ and Fh1−/− mice and labelled Fh1+/−. b, Immunoblots of specified proteins in kidney tissue samples at day 5 post-induction. Five kidney samples are shown. c, Upon loss of Fh1 activity, fumarate cannot enter the enzymatic reaction that converts it into succinate but, instead, can enter a chemical reaction termed Michael addition7 whereby it is chemically added to thiol residues of free cysteine to form 2SC, and of proteins which is considered a metabolic marker of FH activity loss. This reaction can be seen as a “buffering tank” that mop-up the excess of fumarate. Therefore, succinated proteins and 2SC levels increase before an intracellular increase in fumarate is detected. Only when succination is “at saturation” with the 2SC intracellular sinks (or “buffering tanks”) full, fumarate starts to accumulate. Thus, a modest increase in fumarate can still be accompanied by a strong effect. d, Volcano plots showing the differentially expressed genes in Fh1−/− vs Fh1+/+ kidney at day 5 (left) and day 10 (right). e, Deconvolution method on bulk expression data (https://github.com/Danko-Lab/TED and Methods) was applied to determine the cellular composition of mouse kidney tissue at day 5 and day 10 post-induction. Pairwise comparison using Wilcoxon rank sum exact test and Benjamini–Hochberg p-value adjustment were used. f, Immunohistological staining of Fh1fl/fl (top) vs Fh1−/− (bottom) mouse kidney tissue at day 10 post-induction. Left: 2SC staining, right: CD14 staining. Scale bars: 100 μm.
