Extended Data Fig. 1: In vitro evaluation of GLS KO on cellular respiration.
(a) Validation of knockout efficiency in stable transfected OCI-AML3 cells, generated using shRNA control, puro-GAC plasmid, puro-KGA plasmid, puro-D3 (both isoforms) plasmid with puromycin incubation (3 days, 1 µg/ml). The expression of GAC and KGA in OCI-AML3 cells was determined by immunoblotting. Data were quantified in reference to loading control (n = 2 independent experiments, in 3 cell lines tested with similar level of knockdown). (b) Representative Seahorse Mito Stress Test assay graphs obtained from OCI-AML3 cells subjected to inducible knockout of KAG, GAC or GAC/KGA; Results were normalized to viable cell count. (n = 3-4 technical replicates, mean±SD, n = 3 independent experiments). (c) Basal, maximal oxygen consumption rate, OCR-linked ATP production, extracellular acidification rate (ECAR) and global metabolic phenotype (OCR/ECAR) were measured and calculated for OCI-AML3 cells subjected to doxycycline inducible knockout of KGA, GAC or GAC/KAG (mean ± SD, n = 3-4 technical replicates, n = 3 independent experiments, ordinary one-way ANOVA); (d) Representative Seahorse Mito Stress Test assay graphs obtained from four AML cell lines: MV4-11, MOLM13, OCI-AML3, and U937 treated with DMSO or telaglenastat (CB-839) at 1µM, for 12 hours; Results were normalized to viable cell count (n = 3–6 technical replicates, mean±SD, n = 3 independent experiments). (e) Basal, maximal oxygen consumption rate, OCR-linked ATP production, extracellular acidification rate (ECAR) and global metabolic phenotype (OCR/ECAR) were measured and calculated for AML cells as in (D) (n = 3–6 technical replicates, mean ± SD, n = 3 independent experiments, ordinary one-way ANOVA). (f) Seahorse Mito Stress Test assay graphs in OCI-AML3 cells treated with DMSO or BTES at 20µM. Results were normalized to viable cell count (n = 4 technical replicates, mean±SD, n = 3 independent experiments). (g) Mass spectrometry measurement of UMP and AMP in OCI-AML3 cells treated with DMSO or CB-839 1µM for 24 hr (n = 1 biological experiment with n = 4 technical replicates, mean±SD, unpaired t-test).
