Fig 1: MDH1 or MDH2 promoted the proliferation and invasion of primary AT2 cells by promoting glucose uptake. (a, b) RT-PCR and (c, d) WB were used to detect the effect of siRNA and overexpressed plasmid on AT2 cells. (e) MDH1 or MDH2 promoted the glucose uptake of AT2 cells. (f) The proliferation of AT2 cells was dependent on glucose. (g, h) The effect of MDH1 or MDH2 on the proliferation of AT2 cells was measured in (g) 100% high-glucose medium or (h) 100% glucose-free medium via CCK-8. (i) The effect of MDH1 or MDH2 on the proliferation of AT2 cells was measured via EdU assays in 100% high-glucose medium. (j) The invasion of AT2 cells was dependent on glucose. (k, l) The effect of MDH1 or MDH2 on the invasion of AT2 cells was measured in 100% high-glucose medium (k) or 100% glucose-free medium (l) via transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001; NS: no significance.
Fig 2: NTG reduced ATP production in low glucose by inhibiting AMPK activity. (A) Seahorse was used to detect oxygen consumption rate (OCR). A total of 5 × 104 astrocytes were seeded in a well of 24-well plate used in XFe24 Seahorse analyzer. n = 5. Basal OCR and OCR for ATP production and proton leak were calculated with the instruction of Seahorse. (B) Proteins of mitochondrial complexes underwent no significant change after the treatment of NTG, neither in high-glucose nor in low-glucose conditions. (C) Protein level of pAMPK reduced significantly in the low-glucose and NTG group. NTG 2 µg/mL, treated for 4 h. AMPK, AMP-activated protein kinase; CS, citrate synthase; IDH, isocitrate dehydrogenase 2; MDH2, malate dehydrogenase 2. Compared with control, * p < 0.05, ** p < 0.01.
Fig 3: Western blot analysis of total protein. a Effects of H9N2 and H5N1 infection on ATP5F1, ECHS1, HSPA1L and MDH2 total protein expression in A549 cells. b Western blot analysis of BAX and Caspase 3 protein levels in normal A549 cells and H5N1- and H9N2-infected A549 cells. β-actin was used as an internal reference. *, p < 0.05; n = 3
Fig 4: Effects of H9N2 and H5N1 infection on ATP5F1, ECHS1, HSPA1L and MDH2 mitochondrial protein expression in A549 cells.*, p < 0.05 versus H9N2-infected group; n = 3
Fig 5: 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, aKG alpha-ketoglutarate, NOX normoxia, HOX hypoxia
Supplier Page from Abcam for Anti-MDH2 antibody [EPR14882(B)]