Figure. 4

Figure. 4

ADPGK KO B-cells are metabolically toxified. (a)–(d) ADPGK KO and WT cells were analysed for aerobic glycolysis upon stimulation with PMA by measurement of enzyme activities of Hexokinase-2, G6PDH (Glucose 6-phophate dehydrogenase), Pyruvate kinase M2-dimeric (PK-LA) and ATP synthase spectrophotometrically; RT-qPCR for hexokinase-2 (e) for gene expression, and (f) accumulated FITC-NBD-glucose (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose) via flow cytometry for glucose uptake. (g) Serine and Glutamine consumption in Ramos WT and ADPGK KO cells at D2, D7 normalized to their respective unstimulated values. (h) Secreted metabolites in culture media at D2, D7; lactate:pyruvate levels for estimation of Warburg phenotype and Ornithine, NH₃ values for glutamine metabolism, normalized to respective unstimulated conditions. (i) Intracellular amino acids and metabolites measured in ADPGK KO and Ramos WT cell lysates at two and seven days post stimulation with PMA. Data shown are normalized to the respective unstimulated controls. SER- Serine, GLU- Glutamic acid, GLN- Glutamine, CIT- Citrate, NH₃-Ammonia. All graphs are representative of three independent experiments. WT, KO: mean of values from two wild-type and two ADPGK knock-out cell lines, obtained from three independent experiments. y-axis in all graphs, except amino-acid consumption and metabolite secretion, represents respective raw values normalized to unstimulated WT cells. (*p < 0.05; **p < 0.01, ***p < 0.001, calculated using Welch’s t-test for significance).