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. 2022 Aug 29;13(8):748. doi: 10.1038/s41419-022-05169-x

Fig. 3. CCL2 secreted by BC cells manipulates macrophage polarization.

Fig. 3

A Cytokine array of cell supernatants from MDA-MB-231 treated with NC, siEZH2 or EPZ-6438 for 48 h (Human Cytokine Array GS440, RayBiotech, part of the result). B The concentration of CCL2 in the supernatant of MDA-MB-231 treated with siNC, siEZH2 or EPZ-6438, GSK126 for 48 h were detected by ELISA analysis. C The mRNA level of Ccl2 in MDA-MB-231 treated with siNC, siEZH2 or EPZ-6438, GSK126 for 48 h were detected by RT-qPCR. D BMDM were co-cultured with CM from 4T1 treated with siEZH2 and CCL2, or EPZ-6438 and CCR2 antagonist RS 504393 for 48 h. Percentages of CD206+F4/80+ macrophages were analyzed by flow cytometry. BMDM were co-cultured with CM from 4T1 treated with siNC or siCCL2 for 48 h. Percentages of CD206+ F4/80+ macrophages were analyzed by flow cytometry E, the TGF-β level in the supernatant of BMDM were analyzed by ELISA F and protein levels of CCL2, TGF-β, p-STAT3 and p-STAT6 were analyzed by western blot G. H Immunohistochemistry staining of H3K27me3, CD163, F4/80 and CCL2 in the tumor tissue from EPZ-6438 or vehicle treated 4T1 xenograft. Scale bar, 50 μm. Percentages of F4/80+ or CD163+F4/80+ macrophages and the positive staining of CCL2 in IHC was quantified and the shown were representative of replicates. Statistical significance was addressed using unpaired, two‐tailed Student’s t‐test, *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant. The statistical significance was calculated compared to the negative control (NC) in each group. The graphs were shown as means ± SD, n = 3 independent experiments.