Fig. 7: Inhibition of EGFR phosphorylation promote M2 polarization by regulating glutamine metabolism through activation of PPARγ.

A–F RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h, with or without Erlotinib (20 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of M2-related genes Mcr1 (n = 3). B RT-qPCR analysis of mRNA expression of M2-related genes Ym1 (n = 3). C Representative western blot of Arg1. D Flow cytometry analysis showing the level of M2 macrophage-associated markers CD206. E Percentage of CD206-positive RAW264.7 is shown (n = 3). I 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 CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of CD206-positive alveolar macrophage is shown (n = 9). I Mean fluorescence intensity (MFI) is shown (n = 9). J Immunoblot analysis of p-PPARγ (Ser112), t-PPARγ in RAW264.7 cells treated with LPS (1 μg/mL) for 30 min with or without indicated concentration of PD168393 (10 μM) pretreatment for 30 min. K Fluorescence images depicting PPARγ translocation (left panel, scale bar, 50 μm; right panel, scale bar, 5 μm). L–N RAW264.7 cells were treated with LPS (1 μg/mL) for 24 h with or without Erlotinib (10 μM) or Rosiglitazone (Rosi (20 μM) pretreatment. L Cell surface CD206 were analyzed by flow cytometry. M Percentage of CD206-positive RAW264.7 is shown (n = 3). N Mean fluorescence intensity (MFI) is shown (n = 3). O–Q 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. O CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. P Percentage of CD206-positive alveolar macrophage is shown (n = 9). Q Mean fluorescence intensity (MFI) is shown (n = 9). R–V RAW264.7 cells were treated with LPS (1 μg/mL) for 30 min with or without PD168393 (10 μM) pretreatment for 30 min. R Flow cytometry analysis of JC-1 for the detecting the change of mitochondrial membrane potential (ΔΨm) (left panel, JC-1 aggregates; right panel, JC-1 monomers). S The ratio of JC-1 aggregates /JC-1 monomers was calculated as Δψm. T Total cellular ATP level was detected (n = 3). U Immunoblot analysis of IRG1, ATP5A, SDHA, Tubulin as a loading control. V Immunoblot analysis of SDHA and IRG1 in Control or IRG1 silenced RAW264.7 cells, Tubulin as a loading control. The graphs depict mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.