Fig. 2

HIF-1α accumulation in LOX1⁺ PMN-MDSCs from FDNB enhances glycolysis and lactate production. (A) Heatmap showing the expression profile of LOX1⁺ PMN-MDSCs from NNB and FDNB in up-regulated genes. (B) KEGG pathway analysis for the up-regulated genes in LOX1⁺ PMN-MDSCs between NNB and FDNB groups. (C) GO analysis of biological process for the up-regulated genes in LOX1⁺ PMN-MDSCs between NNB and FDNB groups. (E) Representative flow cytometry results (upper) and statistical analysis (lower) of HIF-1α (n = 7), HIF-2α (n = 6), and HIF-1β (n = 6) level in PMN-MDSCs from NNB and FDNB blood. (E, F) Protein levels of HIF-1α, HIF-2α, and HIF-1β in LOX1⁺ PMN-MDSCs from NNB and FDNB were tested by western blotting experiment. (G) mRNA levels of genes involved in glycolysis metabolism (PFKFB2, PFKFB3, PFKFB4, GPI, ALDOA, ALDOC, PKM, LDHA) and lactate transport (SLC16A3) in LOX1⁺ PMN-MDSCs from NNB and FDNB (n = 6). (H) Gene Set Enrichment Analysis (GSEA) plots show that glycolysis was upregulated in FDNB. (I–K) Seahorse analysis of extracellular acidification rate (ECAR) of glycolysis (J) and glycolysis capacity (K) (n = 4). (L) Lactate level in LOX1⁺ PMN-MDSCs between NNB and FDNB groups (n = 5). (M) Quantitative real-time PCR (qRT-PCR) of DNA precipitated via chromatin immunoprecipitation (ChIP) with anti-HIF-1α antibody for binding to S100A8, S100A9, ARG1, LDHA, and SLC16A3 promoters in LOX1⁺ PMN-MDSCs between NNB and FDNB groups (n = 4). Data are pooled from two independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graphs show the mean ± SEM. Statistical significance was determined using a two-tailed unpaired Student’s t-test (E, G, and J-M)