Journal: Cell Death & Disease

Article Title: Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer

doi: 10.1038/s41419-022-05169-x

Figure Lengend Snippet: 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.

Article Snippet: A Cytokine array of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated (AAH-CYT-G3, RayBiotech, 42 human cytokines, part of the results was shown).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Cell Culture, Flow Cytometry, Western Blot, Immunohistochemistry, Staining, Two Tailed Test, Negative Control

Journal: Cell Death & Disease

Article Title: Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer

doi: 10.1038/s41419-022-05169-x

Figure Lengend Snippet: A Differentially expressed miRNAs in MDA-MB-231 EZH2 siRNA or inhibitor-treated cells screened by miRNA-seq (part of the result), aligned according to their expression levels, red showing upregulation in MDA-MB-231 siEZH2 cells. B The miR-124-3p level was analyzed by RT-qPCR in MDA-MB-231 cells treated with EZH2 inhibitors or siRNAs for 48 h. U6 was included as a control. n = 3. C Relationship between has-miR-124 expression level and overall survival of patients with breast cancer ( http://kmplot.com ). D The miR-124-3p and CCL2 level were analyzed by RT-qPCR and western blot in MDA-MB-231 cells treated with miR-124-3p mimics or con-mimics for 48 h. U6 was included as a control. E Western blot showed miR-124-3p mimics repressed CCL2 level induced by EZH2 inhibitors in MDA-MB-231 and 4T1 cells. F The miR-124-3p and Ccl2 level were analyzed by RT-qPCR and western blot in MDA-MB-231 cells treated with miR-124-3p inhibitor or con-inhibitor for 48 h. U6 was included as a control. G Western blot showed miR-124-3p inhibitor induced CCL2 level repressed by EZH2 knockdown in MDA-MB-231 and 4T1 cells. The CCL2 concentration was tested by ELISA in MDA-MB-231 and 4T1 cells treated with EPZ-6438 and miR-124-3p mimics H , or siEZH2 and miR-124-3p inhibitor I . RT-qPCR tested the expression of M2-type markers in THP-1-derived M2 macrophages after being co-cultured with MDA-MB-231 cells treated with EPZ-6438 and miR-124-3p mimics J , or siEZH2 and miR-124-3p inhibitor K . The shown were representative of replicates and the experiments were repeated three times. 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.

Article Snippet: A Cytokine array of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated (AAH-CYT-G3, RayBiotech, 42 human cytokines, part of the results was shown).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Cell Culture, Two Tailed Test, Negative Control

Journal: Cell Death & Disease

Article Title: Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer

doi: 10.1038/s41419-022-05169-x

Figure Lengend Snippet: A The miR-124-3p level was analyzed by RT-qPCR in MDA-MB-231 cells treated with EZH2 siRNA or/and DNMT1 siRNA for 48 h. U6 was included as a control. n = 3. B The miR-124-3p level was analyzed in MDA-MB-231 cells treated with 5 μM 5-Aza-CdR (5-Aza) or/and EPZ-6438 for 48 h. U6 was included as a control. n = 3. C Modified output of MethPrimer program (Li and Dahiya, 2002). Coordinates are given in relation to the transcription start site (TSS). A large CpG island (−1200 to +450 bp, blue region) is evident in the upstream of the gene. Vertical lines indicate relative positions of CpG dinucleotides. D 5-Aza-CdR (5-Aza) and EZH2 siRNA treatment decreased the DNA methylation level of miR-124-3p promoter region (−1164 to −997 and −722 to −605bp) in MDA-MB-231 cells analyzed by MSP. The relative percentages of the methylation were quantified due to the PCR results E . F The CCL2 levels were analyzed by western blot in MDA-MB-231 cells treated with 5-Aza and EZH2 siRNA or 5-Aza and EPZ-6438 for 48 h. G The CCL2 levels were analyzed by western blot in MDA-MB-231 cells treated with siDNMT1 and siEZH2 for 48 h. The shown were representative of replicates and the experiments were repeated three times. In ( A , B and E ), all data were shown as mean values ± SD ( n = 3). 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.

Article Snippet: A Cytokine array of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated (AAH-CYT-G3, RayBiotech, 42 human cytokines, part of the results was shown).

Techniques: Quantitative RT-PCR, Modification, DNA Methylation Assay, Methylation, Western Blot, Two Tailed Test, Negative Control

Journal: Cell Death & Disease

Article Title: Regulation of CCL2 by EZH2 affects tumor-associated macrophages polarization and infiltration in breast cancer

doi: 10.1038/s41419-022-05169-x

Figure Lengend Snippet: A Cytokine array of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated (AAH-CYT-G3, RayBiotech, 42 human cytokines, part of the results was shown). B The IL-10 concentrations of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated were analyzed by ELISA. C Transwell assay for MDA-MB-231 cells treated with EZH2 inhibitors were plated on the upper cell culture inserts, with culture medium alone (NC), or IL-4-activated THP-1 cells (M2 macrophages) plated in the lower chambers, in the presence or absence of an anti-IL10 antibody at 10 ng/ml, or an isotype-matched IgG control (IgG). D Transwell assay for MDA-MB-231 cells treated with EZH2 siRNAs were plated on the upper cell culture inserts, with culture medium alone (NC), or IL-4-activated THP-1 cells (M2 macrophages) plated in the lower chambers, in the presence or absence of IL-10 at 20 ng/ml. E MDA-MB-231 cells were treated with IL-10 at 20 ng/ml for 24 h, and subjected to human phospho-RTK array (R&D, ARY001B). Each kinase is spotted in duplicate. The pairs of dots in each corner (with the exception of the negative control pair at the lower left corner) are positive controls. The upregulated responding kinases were shown in red frame, with the name of the corresponding kinases. MDA-MB-231 cells were treated with or without 5 μM Gefitinib for 48 h before IL-10 stimulation as indicated for 24 h. The migration capacity was analyzed by transwell assay F and the phosphorylation of EGFR, Src and STAT3 and TGF-β level were tested by western blot G . H IHC staining of CD31 in the tumor tissue from EPZ-6438 or vehicle treated PDX6305 model and BT549 control or EZH2 sgRNA xenografts. Scale bar, 50 μm. The positive area of CD31 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. I Model describing that EZH2 in BC promotes tumor progression and metastasis by increasing CCL2-dependent recruitment and polarization of M2 TAMs, which reciprocally secrete IL-10, VEGF and other cytokines to activate EGFR signaling in cancer cells (the lung is created from BioRender.com).

Article Snippet: A Cytokine array of cell supernatant from THP-1 derived M2 macrophages co-cultured in different MDA-MB-231 CM as indicated (AAH-CYT-G3, RayBiotech, 42 human cytokines, part of the results was shown).

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Transwell Assay, Negative Control, Migration, Western Blot, Immunohistochemistry, Two Tailed Test