Fig. 4: Metabolic rewiring upon Rnf20 haploinsufficiency.
From: RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α
a KEGG pathway analysis of genes upregulated in Rnf20 + /- MLE12 cells compared to controls. Significance is presented as -Log10 (p-values) from KEGG pathway analysis using DAVID Bioinformatics Resources. b Western blot analysis for HIF1α in control and Rnf20 + /- MLE12 cells under normoxic or hypoxic conditions. c Immunostaining for HIF1α (left panels) and HIF1α luciferase reporter activity (right panel) in control and Rnf20 + /- MLE12 cells (n = 3) under normoxic conditions. Scale bars, 20 µm. d Targeted LC-MS/MS-based metabolomics of control and Rnf20 + /- MLE12 cells (n = 6). The Y-axis represents the relative concentration (μM) per 1 million cells. Metabolic enzymes that show changed expression in Rnf20 + /- MLE12 cells are shown in red. The heatmap in the middle shows the gene expression changes in control and Rnf20 + /- MLE12 cells. e Extracellular acidification rate (ECAR) of control and Rnf20 + /- MLE12 cells (n = 3) recorded following the addition of glucose, oligomycin, and 2-DG. f, g 2-DG uptake (f) and concentration of lactate in the supernatant (g) of control and Rnf20 + /- MLE12 cells (n = 3). h qPCR analysis of key glycolytic enzymes and hypoxia-regulated genes in control (n = 8) and Rnf20 + /- (n = 8) lungs. i, j Immunostaining for HIF1α (i), and immunohistochemistry for GLUT1 and LDHA in lung tissues sections (j) of wild-type and Rnf20 + /- mice. Scale bars in (i) 100 µm; in (j) 200 µm. k Concentration of glucose-6-phosphate and fructose-6-phosphate in lung homogenates (top and middle panels, n = 10 each), and lactate in the serum (bottom panel) of wild-type (n = 3) and Rnf20 + /- (n = 4) mice. Statistical analysis between two groups in (c, d, e, f, g, h, k) was performed using an unpaired two-tailed Student’s t-test. Data are shown as mean ± SEM. ‘n’ indicates biological replicates.
