Fig. 5: PGK1 crotonylation is indispensable for coordinating mitochondrial pyruvate metabolism and glycolysis. | Nature Communications

Fig. 5: PGK1 crotonylation is indispensable for coordinating mitochondrial pyruvate metabolism and glycolysis.

From: Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle

Fig. 5

a The enzymatic activity of Flag-tagged PGK1 WT, K131R, K156R, K220R, or 3KR purified from HEK293T cells were measured. b MDA-MB231 cells expressing Flag-tagged PGK1 WT or 3KR were stimulated with or without hypoxia for 12 h and stained with anti-Flag antibody and Mito-Tracker. Nuclei were stained with DAPI. c Co-IP assays were performed using HEK293T cells transfected with Flag-tagged PGK1 WT or 3KR. d PDHK1 and PDH phosphorylation was detected by immunoblotting in PGK1-deleted MDA-MB231 cells expressing sgRNA-resistant PGK1 (rPGK1) WT or 3KR, with or without shRNA targeting ECHS1. e PDH phosphorylation was detected by immunoblotting in PGK1-deleted MDA-MB231 cells expressing sgRNA-resistant PGK1 (rPGK1) WT or 3KR, with or without PDHK1 depletion. f MDA-MB231 cells with or without ECHS1 depletion were transfected with Flag-tagged PGK1 and cultured under normoxia or hypoxia for 12 h, followed by Co-IP assays. g MDA-MB231 cells with or without ECHS1 depletion were cultured under normoxia or hypoxia for 12 h, followed by immunoblotting with the indicated antibodies. h, i The oxygen consumption rate (OCR, h) and the extracellular acidification rate (ECAR, i) in MDA-MB231 cells with endogenous PGK1 deletion and reconstituted expression of rPGK1 WT or rPGK1 3KR, with or without ECHS1 depletion. j Heat map showing the differential metabolites between MDA-MB231/rPGK1 WT cells and MDA-MB231/rPGK1 3KR cells with or without ECHS1 depletion. Each group had 3 biologically independent samples. k The relative levels of [U-13C3]-Pyruvate derived m + 2 acetyl-CoA in MDA-MB231/rPGK1 WT cells and MDA-MB231/rPGK1 3KR cells with or without ECHS1 depletion. l The levels of mitochondrial ROS were measured using a 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. a, h–l Data are shown as the mean ± SD, n = 3 (a, j, k) or 4 (h, i, l) biologically independent samples (unpaired two-tailed Student’s t test). All experimental data were verified in at least two ( j, k) or three (a–i, l) independent experiments. Source data are provided as a Source Data file.

Back to article page