Extended Data Fig. 3: ACO2 and ACL disruption in embryonic stem cells.
From: A non-canonical tricarboxylic acid cycle underlies cellular identity
a, Fractional M+2 enrichment of citrate and malate in mouse ES cells cultured in medium containing [U-13C]glucose. b, Fractional enrichment of malate M+2 relative to citrate M+2 (mal+2/cit+2) derived from [U-13C]glucose in ES cells following treatment with vehicle or 50 μM BMS-303141 (ACLi) for 24 h. c, d, Immunoblot of clonal mouse ES cells in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) and Acly (ACLY-1 and ACLY-2) (c) or Aco2 (ACO2-1 and ACO2-2) (d). e, f, Assessment of the [U-13C]glucose-derived mal+2/cit+2 ratio (e) or steady-state levels of TCA cycle metabolites represented as the fold change (expressed in log2) relative to Ctrl (f) in control and Aco2-edited ES cells. g, h, j, k, Fractional M+1 enrichment of NADH (g), lactate (h), fumarate (j) and succinate (k) in control and Acly-edited ES cells cultured in medium containing [4-2H]glucose. i, Schematic depicting deuterium transfer from [4-2H]glucose first onto malate in the cytoplasm then onto TCA cycle metabolites in the mitochondria. l, Quantification of the lactate over pyruvate ratio in control and Acly-edited ES cells. m, The baseline oxygen consumption rate (OCR) in control and Acly-edited ES cells normalized to protein content. Twelve technical replicates were averaged for each of three independent experiments. Data are mean ± SD, n = 3 independent replicates unless otherwise noted. Significance was assessed using unpaired two-tailed Student’s t-test (a, b) or in comparison to control cells by one-way ANOVA with Sidak’s multiple comparisons post-test for all other panels.
