Fig. 6: Inhibition of EGFR phosphorylation suppresses glycolysis-dependent M1 polarization in macrophages.

A–F BMDMs were treated with LPS (1 μg/mL) for 24 h with or without PD168393 (10 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of IL-1β (n = 3). B RT-qPCR analysis of mRNA expression of iNOS (n = 3). C Western blot was used to detect the expression of iNOS. D iNOS expression on the surface of BMDM was analyzed by flow cytometry. E Percentage of iNOS -positive BMDM is shown (n = 3). F Mean fluorescence intensity (MFI) is shown (n = 3). G–I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G iNOS expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of iNOS-positive alveolar macrophage is shown (n = 9). I Mean fluorescence intensity (MFI) is shown (n = 9). J Cluster analysis of differentially expressed metabolites in RAW264.7 measured after treated with LPS (1 μg/mL) for 30 min with or without PD168393 (PD 10 μM) pretreatment for 30 min, when compared with control. Blue and red indicates down-or upregulation, respectively (n = 3 samples of each condition). K Schematic illustrating the metabolites that are decreased (blue) in PD168393 (10 μM) RAW264.7 cells at 24 h after LPS stimulation. L PD168393 (10 μM) pretreatment RAW264.7 cells exhibited a ~2 -fold decrease in lactate levels compared with LPS group (n = 3). M Representative western blots of HIF1-a, p-PKM2, PKM2, LDHA expression in RAW264.7 cells. The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.