Extended Data Fig. 3: Expression and activity of in trans IDH2 second-site mutations in haematopoietic cells.
From: Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations
a, Allele-specific quantitative PCR (qPCR) showing similar expression of constructs in Ba/F3 cells co-transduced with IDH2R140Q (RQ) plus IDH2WT (WT), IDH2Q316E (QE), or IDH2I319M (IM) in trans. Control from IDH2WT human cell line (293T). Data are mean ± s.e.m. for triplicate reactions. b, Intracellular 2HG levels in Ba/F3 cells co-expressing RQ plus WT, QE or IM in trans and treated with vehicle or increasing doses of AG-221 (1 nM, 10 nM, 100 nM, 1 μM or 10 μM). Data are mean ± s.e.m. for triplicate cultures. c, Western blot showing IDH2 protein levels in primary HSPCs from Idh2R140Q/Flt3ITD mice transduced with WT, QE or IM and untransduced control cells for comparison. GAPDH serves as a loading control. The same membrane was stripped and reprobed for western blots. d, Intracellular 2HG levels in primary HSPCs from Idh2R140Q/Flt3ITD mice transduced with WT, QE or IM and collected from the first passage of methylcellulose cultures containing AG-221 at 50 nM. Data for are mean ± s.e.m. for triplicate cultures. e, Flow cytometry gating strategy for Fig. 3i. SSC-A, side scatter area; FSC-A, forward scatter area. DAPI is a viability dye. mCherry identifies retrovirally transduced cells. Results are representative of ≥2 (a–d) or 1 (e) independent experiments. For gel source data, see Supplementary Fig. 1.
