*2.14. Bisulfite Sequencing and CpG Methylation Status*

Genomic DNA was extracted using the DNeasy Tissue kit (QIAGEN, MD, USA). The EZ DNA Methylation Kit (Zymo Research, CA, USA) was used for sodium bisulfite

conversion, according to the manufacturer's protocol. Primers spanning two promoters of the Keap1 gene were designed using Methyl Primer Express (Thermo Scientific). Promoter 1 Forward: 50 - GAGTTTTGGYGGGGAATT-30 ; Reverse: 50 -CCCTACCRCCTAAAACCAA-30 . Bisulfite-modified DNA (100 ng) was amplified in a PCR mix containing 0.4 µM of forward and reverse primer, HotStarTaq Master Mix Kit (Qiagen, Germany: 203445). Methylation status analysis was performed by Quantification Tool for Methylation Analysis (QUMA) software.

## *2.15. Statistical Analysis*

One-way analysis of variance (ANOVA), followed by Tukey's post hoc multiple comparisons tests by Prism software (GraphPad, San Diego, CA, USA), was used to analyze statistical significance. All the data values are presented as mean ± SE, reflecting the minimum of three independent determinations. Statistical significance was determined by comparing the treatments with untreated controls, and the significant differences are indicated as \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.
