Fig. 3
PHD3 regulates glucose metabolism of ccRCC cells. a PECA analysis results with log2 fold change and FDR. H hypoxic, N normoxic. Upregulated proteins marked by red and downregulated by blue. b Illustration of the glycolytic pathway. Red ovals represent upregulated and blue ovals downregulated proteins in response to PHD3 knockdown in 786-O cells according to PECA analysis. Clear ovals represent glycolytic enzymes not affected by PHD3 depletion based on LC-MS/MS analysis. c Extracellular lactate concentration normalized to cell count of 786-O and RCC4 cells in normoxia and hypoxia shows a decrease with PHD3 silencing. Quantification of three (786-O) or two (RCC4) biological replicates, mean ± SEM (*p < 0.05, n.s. not significant). d pH measured from 786-O cell culture medium in normoxia and hypoxia shows lower level of extracellular acidification with PHD3 depletion. Hydronium ion concentration was normalized to cell count and converted to pH. Quantification of three biological replicates, mean ± SEM (**p < 0.01, n.s. not significant). e Oxygen consumption rate (OCR) studied with Seahorse XFp Analyser shows an increase in basal OCR and in maximal OCR with PHD3 depletion. Data presented as mean ± SEM, n = 3 (**p < 0.01). f Basal glycolysis function measured with Seahorse XFp Analyser shows a decrease with PHD3 depletion. Mean ± SEM, n = 3. Key: GLUT1 solute carrier family 2, facilitated glucose transporter member 1, HK1 hexokinase 1, G6PD glucose 6-phosphate 1-dehydrogenase, NADPH nicotinamide adenine dinucleotide phosphate, TALDO1 transaldolase, GPI glucose-6-phosphate isomerase, PFKP 6-phosphofructokinase, GFPT glutamine-fructose-6-phosphate aminotransferase, F6P fructose 6-phosphate, F1,6BP fructose 1,6-bisphosphate, ALDO fructose-bisphosphate aldolase, TPI1 triosephosphate isomerase, G3P glyceraldehyde 3-phosphate, GAPDH glyceraldehyde phosphate dehydrogenase, PGK phosphoglycerate kinase, PGAM1 phosphoglyserate mutase 1, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, ENO1 Alpha-enolase, PEP phosphoenolpyruvate, PKM pyruvate kinase isoenzyme M, LDHA l-Lactate dehydrogenase A chain, LDHB l-Lactate dehydrogenase B chain, MDH2 malate dehydrogenase, NADH nicotinamide adenine dinucleotide, IDH1 isocitrate dehydrogenase, αKG alpha-ketoglutarate, NOX normoxia, HOX hypoxia