Figure 5. Treatment with demethylating agents restores EGFR induction under hypoxic conditions for cell lines with methylated HIF-binding regions of EGFR.

A-B. MS-HRM assay was used to evaluate the methylation status of MDA-MB-231 cells treated with 500nM AZA, 250nM DAC or DMSO for 3 consecutive days followed by 10 days of culture in the absence of drug. (A) Methylation percentage after standardizing to MCF-7 and normalizing by MDA-MB-231(MDA-231) levels. (B) MS-HRM analysis using UAnalyze software. C-D. A ChIP assay was performed to assess the enrichment of EGFR in MDA-MB-231 cells treated with 250nM DAC or DMSO for 3 consecutive days followed by 8 days of culture in the absence of drug and exposed to 20% or 1% O₂ for 5 h using antibodies against (C) HIF-1α or (D) HIF-2α (mean ± SEM, n = 3); *P < 0.05 versus MDA-MB-231 cells treated with DMSO (control) at 20% O₂ (two-way ANOVA with Bonferroni posttest).E-F. EGFR mRNA levels measured by qPCR in MDA-MB-231 (MDA-231) cells treated with (E) 500nM of AZA or (F) 250nM of DAC for 3 consecutive days followed by 6, 8, or 10 days of culture in the absence of drug. Cells were exposed to 20% or 1% O₂ for the last 24 h of the experiment (mean ± SEM, n = 3); **** P < 0.0001 versus MDA-MB-231 cells treated with equivalent concentration of DMSO (control) at 20% O₂ (two-way ANOVA with Bonferroni posttest). G-H. Immunoblot assays were performed using lysates prepared from MDA-MB-231 cells treated with 500nM AZA (G) or 250nM DAC (H) for 3 consecutive days followed by 10 days of culture in the absence of drug. Cells were exposed to 20% or 1% O₂ for the last 48 h of the experiment.