Fig 2. Genetic deletion of PKM2 abrogated the protective effect of scFv 5.

Reconstitution of PKM2-deficient MEFs with PKM2 WT or PKM2 (K422R), but not PKM1 or PKM2 (K240M), restored cell death protection. The PKM2-activating compound TEPP-46 enhanced the clonogenic survival effect of IB5. A. Viability assays with reconstituted cells. WT MEFs, PKM2-deficient MEFs, or PKM2-deficient MEFs reconstituted with WT or mutant PKM2 cDNA were infected or not with IB5, then later transfected with BimS expression plasmid. Surviving cells were counted 48 h afterwards. Note that only WT cells or PKM2-deficient MEFs reconstituted with WT PKM2 exhibited cytoprotective activity of IB5. B. IB5 produced clonogenic survival despite BimS expression. Control or IB5-expressing cells were transfected with BimS cDNA, and after 5 d, the plates were fixed with 6.0% glutaraldehyde and stained with 0.5% crystal violet. Top: example crystal violet-stained plate; bottom: average colony counts from three independent experiments, ± SEM. C. IB5-induced clonogenic rescue of 293T cells from BimS was enhanced by treatment with TEPP-46. Control 293T or IB5-expressing cells were incubated with or without 27 g/ml TEPP-46 for 3 h, then transfected with BimS cDNA in a further 24-h incubation also including TEPP-46 or vehicle. The plates were fixed and stained with crystal violet after 1 week. Left: examples of crystal violet-stained plates; right: as cells did not typically grow as discrete colonies, we quantified the total area of colonies (a measure of the total number of proliferating cells) formed in each well using ImageJ software [78]; mean ± SEM are shown from three independent experiments. Note: underlying data are included in corresponding tabs in the accompanying supplemental Excel file, S1 Data. 293T, HEK293T; IB, intrabody; MEF, Mouse Embryonic Fibroblast; PKM1/M2, pyruvate kinase isoform M1/M2; scFv, single-chain variable fragment; WT, wild-type.