Figure 2.
Imbalanced Glycolytic and Oxidative Metabolisms in VHL-Deficient B Cells
(A) Scatterplot of gene expression of microarray analysis of VHL-deficient and WT B cells. Genes significantly upregulated (fold-change > 1.5, p < 0.05) and downregulated (fold-change < −1.5, p < 0.05) were shown in red and blue, respectively.
(B) Gene set enrichment analysis (GSEA) enrichment curve of HIF-1α target genes in VHL-deficient B cells (enrichment score [ES] = 0.927, normalized ES = 2.41, false discovery rate q value < 0.01). Vertical black lines indicate HIF-1α target genes detected by microarray analysis.
(C) Heatmap depicting log2 fold change of expression of HIF-1α and HIF-2α target genes in VHL-deficient versus WT B cells. Genes that were identified as differentially expressed with statistical significance were indicated with a black border.
(D) Schematic of glycolytic pathway.
(E) Immunoblotting of glycolytic enzymes in VHL-deficient and WT B cells. β-Actin was used as the loading control.
(F and G) Analysis of extracellular acidification rate (ECAR) in VHL-deficient (blue) and WT (red) B cells. Real-time changes in ECAR of B cells with the addition of glucose (Glc) (10 mM), oligomycin (Oligo) (1.0 μM), and 2-deoxy-d-glucose (2-DG) (50 mM) was measured (F). Basal glycolysis was quantified as the difference of ECAR before and after addition of glucose (G).
(H) Quantification of lactate produced by B cells. The amount of lactate in the supernatant of equal number of VHL-deficient and WT B cells cultured for 1 h was quantified.
(I) Oxygen consumption rate (OCR) in VHL-deficient and WT B cells. OCR of B cells with the addition of oligomycin (Oligo) (1.0 μM), FCCP (1.0 μM), and rotenone plus antimycin (Rot/AA) (0.5 μM for each) was measured in real time. Spare respiratory capacity (SRC) is indicated as the difference between basal OCR and maximum OCR after addition of FCCP.
(J) Quantification of basal OCR (leaf panel) and SRC (right panel) in VHL-deficient and WT B cells. Bar graphs represent mean ± SD (n = 5 for G and J; n = 3 for H).
Data shown are representative of more than 3 experiments (E, n = 3; F and I, n = 5). For F and I, each data point represents mean ± SD, n = 5. ***p < 0.001 (Student's t test). See also Figures S2 and S7.