Extended Data Fig. 8: Increased L,D-2HG levels and 5mC in Dnmt3aR882H/+ upon Slc25a1 knockout.
From: Mitochondrial metabolism sustains DNMT3A-R882-mutant clonal haematopoiesis
(a) Schematic representation of experimental strategy. (b) The levels of 2HG in Slc25a1 ko/control WT and Dnmt3aR882H/+ cells. Mean ± SD is shown, n = 3 biological replicates per group, P by one-way ANOVA. (c) Chromatogram indicating L and D-2HG in exemplary WT sample upon knockout of Slc25a1. L-2HG and D-2HG standards are presented below. Similar data were observed for n = 3 biological replicates. (d) Levels of L-2HG and D-2HG in each sample by LC-MS. Mean ± SD is shown, n = 3 biological replicates per group, P by two-way ANOVA. (e) DNA methylation and hydroxymethylation in WT and Dnmt3aR882H/+ HSPCs by dot blot. (f) Quantification of 5mC, n = 5 biological replicates per group, and (g) 5hmC levels in Dnmt3aR882H/+ relatively to WT. n = 5 for WT and n = 6 biological replicates for Dnmt3aR882H/+. (h) Dot blot and quantification of 5mC levels in WT Slc25a1 null cells compared to WT Empty controls, and (i) Dnmt3aR882H/+ Slc25a1 null cells compared to Dnmt3aR882H/+ Empty controls. (j) 5hmC levels in WT and (k) Dnmt3aR882H/+ compared to Empty controls. (f-k) Mean ± SD is shown, in (j) n = 5 biological replicates for WT Empty, and in remining groups in (h-k) n = 6 biological replicates, P by two-sided t-test. Schematic in a was created using BioRender (https://biorender.com).
