Fig. 3

Metformin reversed SAHA-induced feedback activation by inhibiting histone acetylation on FGFR4. A The Venn diagram displayed the intersection of upregulated genes in the SAHA-treated group, downregulated genes in the Metformin-treated group, downregulated genes in the SAHA + Metformin combination group, and membrane receptor genes. B GSEA analysis was performed on the differential expression results between the SAHA-treated group and the control group. C Immunoblotting showed the change in FGFR4 and STAT3 phosphorylation. MDA-MB-231 cells pretreated with JQ1 for 24 h were exposed to SAHA for a further 12 h. FGFR4 and STAT3 phosphorylation changes were detected by immunoblotting. All bands were quantified from experiments repeated three times. D Histone acetylation on the FGFR4 promoter. (Left) MDA-MB-231 cells were treated with SAHA (5 μM) for 12 h before being subjected to ChIP assay using anti-acetylhistone H3K9 (Ac-H3K9) antibody followed by qPCR analysis using primers targeting the indicated FGFR4 promoter region. (Middle) BRD4 enrichment on the FGFR4 promoter. MDA-MB-231 cells were treated with SAHA (5 μM) for 12 h before being subjected to ChIP assay using anti-BRD4 antibody. qPCR analysis was performed using primers targeting the indicated FGFR4 promoter region. (Right) MDA-MB-231 cells were treated with Metformin (20 mM) for 48 h before being subjected to ChIP assay using anti-acetylhistone H3K9 (Ac-H3K9) antibody followed by qPCR analysis using primers targeting the indicated FGFR4 promoter region. All experiments were performed in triplicate. Error bars represent means ± SD from triplicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E FGFR4 mRNA level changes. MDA-MB-231, BT-549, and HCC1806 cells were treated with SAHA (5 μM) for 24 h or metformin (20 mM) for 48 h. Samples were analyzed by qPCR assay. Error bars represent means ± SD from triplicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F Immunoblotting showed the change in FGFR4 and STAT3 phosphorylation. (Left) MDA-MB-231 cells were treated with indicated SAHA (0, 5, 10 μM) for 12 h. (Middle) MDA-MB-231 cells were treated with indicated metformin (0, 10, 20, 40 mM) for 48 h. (Right) MDA-MB-231 cells pretreated with metformin (20 mM) for 36 h were exposed to SAHA (5 μM) for further hours. FGFR4 and STAT3 phosphorylation changes were detected by immunoblotting. All bands were quantified from experiments repeated three times