Fig 2: Co-targeting BRD4 and RAC1 targets c-MYC-G9a-FTH1 axis and induces autophagy. The expression of c-MYC, G9a and FTH1 was examined in MCF-7, MDA-MB- 231, SKBR3 and JIMT-1 cells treated with (A) JQ1 (2 uM) and/or NSC (30 uM) or combination for 72 h; β-actin was used as a negative loading control. Expression status of (B) LC3BI/II was evaluated in MCF-7, MDA-MB-231, SKBR3 and/or JIMT-1 cells at 72 h following treatment with JQ1 (2 uM) and/or NSC (30 uM) for 72 h. β-actin was used as a negative loading control.

Fig 3: FTH1 knockdown promotes mammosphere formation efficiency and cell migration in BCa cells. (A) Mammosphere formation was evaluated in MDA‐MB‐231, JIMT1, SKBR3, and MCF‐7 cells transfected with control or FTH1‐specific siRNA and cultured in ultra‐low attachment plates for 7 days post‐transfection (20× magnification). (B) Error bars represent the mean ± SD of mammosphere numbers based on four independent experiments. (C) Wound‐healing assay was performed in MCF‐7 cells transfected with control or FTH1‐specific siRNA for 72 h post‐transfection (20× magnification); scale bar represents 200 μm. (D) Mean ± SD of wound‐healing area at 24 h postwound administration based on five independent experiments. Statistical analysis was performed using the Student t‐test; *P < 0.05; # no significant difference.

Fig 4: Reduced c‐MYC and G9a expression associates with enhanced cell sensitivity to chemotherapy. Viability of MCF‐7 cells silenced for FTH1 24 h prior to treatment with increasing concentrations of (A) DFO, (B) 5‐FU, (C) vitamin C, (D) doxorubicin, or (E) JQ1. (A‐E) Error bars show the mean ± SD from four replicated experiments. Cell viability of MCF‐7 cells silenced for G9a 24 h prior to treatment with increasing concentrations of (F) SAHA or (G) 5‐FU or silenced for c‐MYC 24 h prior to treatment with increasing concentrations of (H) SAHA or (I) 5‐FU. MCF‐7 cells silenced for c‐MYC 24 h prior to treatment with increasing concentrations of SAHA or 5‐FU. Cell viability was assessed by CV staining at 72 h post‐treatment. (F, I) Error bars show the mean ± SD from four replicated experiments. One‐way ANOVA was performed to test for statistical significance among multiple groups; *P < 0.05, **P < 0.005, # not significant.

Fig 5: Correlation of FTH1, MYC, and G9a with key IRGs in BCa samples. mRNA coexpression analysis of (A) c‐MYC, (B) G9a, or (C) FTH1 vs genes that encode the TfR1 gene (TFRC), STEAP3, hepcidin (HAMP), FPN (SLC40A1), hephaestin (HEPH), and FTL chain using TCGA BCa dataset (1084 patient samples; http://www.cbioportal.org); R and P values were generated using Pearson statistic.