Fig. 2
IL-6 upregulated the expression of KDM2A in mammary fibroblasts through the STAT3/NFκB p50 axis. A ChIP assay was performed to pull down the STAT3 proteins-chromatin complexes in IL-6-treated mammary fibroblasts using anti-STAT3 antibody. STAT4 and NFκB p50 binding motifs on the KDM2A promoter region were amplified by PCR. Each experiment was performed in triplicate and repeated three times independently. Data are expressed as the fold change relative to non-treated control cells. The upper region of the histogram shows the putative STAT4 and NFκB p50 binding motif on the KDM2A promoter region. B RMF-EG cells were incubated with HS-578 T CM for 48 h in the presence and absence of JSH-23. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. C RMF-EG cells were treated IL-6 for 48 h in the presence and absence of 30 μM JSH-23. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. D Ectopic pSTAT3-expressing RMF-EG cells were treated with or without JSH-23 for 48 h. KDM2A protein level was analyzed by Western blotting analysis. E Immunoprecipitation assay was performed in IL-6-treated RMF-EG cells. STAT3 bound protein complexes were pulled down using anti-STAT3 antibody and analyzed by immunoblotting with anti-NFκB p65 and anti-NFκB p50 antibodies. Differences were found to be statistically significant at *p < 0.05, ** p < 0.01, and *** p < 0.001