Figure 7. Overexpression of CCL1 rescues the inhibited recruitment of Tregs induced by Sox2 silencing and promotes tumor growth in vivo.
A Western blotting results of CCL1, Sox2, and Ki67 proteins in EO771, 4T1, and 4TO7 cells. B. CCK8 assay for the proliferation status of 4TO7 and 4T1 cells in vitro, data are presented as the mean + SD, n = 5–6. C. Growth curves of breast tumor xenografts in BALB/c mice, n = 6–9 mice. 1×105 4TO7 cells or 5×104 4T1 cells were inoculated to the fourth mammary gland of each female BALB/c mouse at 6–8 week old, respectively. Statistical significances for the tumor volume between different groups are determined at 19th day post graft for 4TO7, and at 29th day post graft for 4T1. D. Western blotting results of nuclear p65 in 4TO7, 4T1, and EO771 cells. E. Western blotting showing expression changes of CCL1 and nuclear p65 proteins in 4TO7 cells after treatment with 10 μM PDTC, which is the inhibitor of NFκB signaling pathway. F. Dual luciferase method was used to analyze the activity of NFκB signaling in EO771 cells, n = 6–9. G. Western blotting showing the expression of nuclear H3K27Me3 in EO771, 4T1, and 4TO7 cells. H. Quantitative ChIP assay showing the binding changes of H3K27Me3 on the promoter regions of Ccl1 and p65 upon Sox2 overexpression in 4TO7 cells. The approximate distance from the transcriptional start site (TSS) is indicated, n = 3. I. Schematic summary of our proposed model: Tregs enhance the stemness of breast cancer cells. Reciprocally, the CSCs recruit Tregs through SOX2-NFκB-CCL1 signaling. ns, not significant; *, p < .05; **, p < .01. Abbreviations: DMSO, dimethyl sulfoxide; PDTC, pyrrolidine dithiocarbamate.