Fig. 6: The IRF4 promoter is hypermethylated in BC patients and correlates with poor overall survival.

a Box plot showing the methylation status of IRF4 in BC patients using the mean aggregation of all 16 CpGs referenced within the SMARTapp obtained from the TCGA project. The outer limits of the box represent the 25th (lower quartile) and 75th percentile (upper quartile) with the median value shown inside. Whiskers extend to the lowest and highest values. ****p < 0.0001; Student’s t test. b Schematic representation of the IRF4 promoter containing ten CpG sites identified by SMARTapp found within three CpG islands (blue bars). The corresponding relative mean methylation levels for each of the 10 promoter-specific CpG sites in normal tissue and BC tumor samples (M-value) are shown. The associated p values were calculated using the Student’s t test. The pairs of primers used for MS-qPCR analysis in i–k are represented as F1-R1 for cg26433102, F2-R2 for cg06392169, and F3-R3 for cg21277995. c–e Kaplan–Meier survival curves derived from the SMARTapp illustrating the correlation between the methylation status (M-value) of the IRF4-associated CpG sites cg26433102 (c), cg06392169 (d), and cg21277995 (e) with BC patient overall survival. f–h Spearman correlation curves obtained from the SMARTapp showing the association between the methylation status (M-value) of the IRF4-associated CpG sites cg26433102 (f), cg06392169 (g), and cg21277995 (h) and IRF4 gene-level expression in BC patients (n = 853). i–k Relative methylation levels of the IRF4-associated CpG sites cg26433102 (i), cg06392169 (j), and cg21277995 (k) after treatment with C29 (5 μM), 5-azadC (3 μM) or a combination of both drugs for 7 days in SKBR3 cells. Data presented in (i–k) show means ± SEM. **p < 0.01, ***p < 0.001; Student’s t test.