Fig 1: Functional validation of M-CSF-induced macrophage differentiation from PαS and human MSCs(A) Visual summary for blocking M-CSF-induced macrophage differentiation. PαS cells isolated from the bone marrow and cultured in M-CSF-containing medium with or without CSF1R-blocking antibody or small molecule inhibitor (GW2580). Representative immunofluorescence images show CD206 (green), CD45 (red), and CD68 (gray) expression. Scale bars, 200 μm.(B) Experiment process for M-CSF injection. M-CSF was injected into the tail vein 7 days and again 3 days before collecting PαS cells, and the PαS cell yield was analyzed by flow cytometry.(C) CD45− TER119-cells after M-CSF tail vein injection (PBS, n = 5, MCSF, n = 5) (ns, not significant, Student’s t tests).(D) PαS cells in the bone in response to M-CSF injection (PBS, n = 5, MCSF, n = 5) (∗p < 0.05, Student’s t tests).(E) CSF1R expressing PαS cells in response to M-CSF injection (PBS, n = 5, MCSF, n = 5) (ns, not significant, Student’s t tests).(F) Experimental setup for human cell analysis. CD90+ CD271+ human MSCs (huMSCs) and human bone marrow cells (huBMCs) were cultured under DMEM or M-CSF-containing RPMI (RM) conditions.(G) Expression of CSF1R in freshly isolated huMSCs from the BM (CD271+ CD90+ cells).(H) Immunofluorescence staining of huBMCs and huMSCs after culture in DMEM or RM. Cells were isolated from the bone marrow and stained for CD206 (green), CD45 (red), and CD68 (gray). All results were confirmed in three independent experiments. Scale bars, 200 μm.
Fig 2: Mechanism underlying the modulating effect of ZEB2 on cytokine secretion. (A) The binding motif of ZEB2 quoted from previous research[35]; (B) Schematic illustration showing that the binding motif of ZEB2 and its palindromic sequence could match several regions of the potential promoter sequences of CSF-1, IL4, and CCL8; (C) Relative luciferase activity in 293T cells transfected with plasmids containing CSF-1 or IL4 or CCL8 promoter (GV534-Promoter-OE), a plasmid expressing ZEB2 (GV712-ZEB2-OE), and the corresponding empty vector; (D) Schematic illustration indicating that, in a PI3K-Akt-dependent manner, ZEB2 is upregulated following EGFR-TKI resistance. This upregulation promotes M2 polarizaiton and inhibits M1 polarization of TAMs by increasing the secretion of CSF-1 and TGF-β1 in NSCLC. ***P < 0.0005.
Fig 3: ZEB2 promotes M2 polarization and impedes M1 polarization of TAMs by elevating the secretion of CSF-1 and TGF-β1. (A) Heatmap demonstrating the relationships between 17 most highly upregulated DEGs after EGFR-TKI resistance in HCC827 and HCC4006 cells and cytokines that were proved to be associated with macrophage polarization; (B) A GSEA was conducted with the ZEB2-related genes in the TCGA LUAD dataset using the LinkedOmics online platform. The results of the KEGG pathway enrichment analysis showed that the chemokine signaling pathway and leukocyte transendothelial migration were among the upregulated pathways; (C) qRT-PCR was conducted to determine the alterations of cytokines associated with macrophage polarization after EGFR-TKI resistance in PC9 and HCC827 cells. Data represent results from three independent experiments; (D) Results of ELISA and MSD electrochemiluminescence of PC9 and HCC827 cells before and after EGFR-TKI resistance. The concentrations of TGF-β1 and CSF-1 were detected using ELISA, while the concentrations of CCL8, IL4, and CXCL9 were detected using MSD electrochemiluminescence. Data represent results from three independent experiments; (E) qRT-PCR analysis revealed that ZEB2 knockdown could reverse the alterations of CSF-1, TGF-β1, CCL8, IL4, and CXCL9 in PC9-GR and HCC827-GR cells. Data represent results from three independent experiments; (F) Results of ELISA and MSD electrochemiluminescence of PC9-GR and HCC827-GR cells with or without ZEB2 knockdown. The concentrations of TGF-β1 and CSF-1 were detected using ELISA, while the concentrations of CCL8, IL4, and CXCL9 were detected using MSD electrochemiluminescence. Data represent results from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.
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