Fig. 4

Absolute methylation and relative expression levels for FKBP1B and PAX9 and the effect of overexpression of candidate genes on cell sensitivity to CDDP in the OVCAR3 cell line. A, B Representation of methylated and unmethylated molecules assessed through qMSP (quantitative methylation-specific PCR). The methylation rate obtained from three replicates for each type of evaluated sample is shown. Assays were performed in all experimental conditions: S, R and RT. S, sensitive; R, resistant; RT, resistant treated with epigenetic reactivation drugs (5-Aza and TSA). Each trial was repeated twice, and a non-neoplastic ovary sample was included as the control. C, D The expression levels of each candidate gene assessed by qRT-PCR were normalized using GAPDH as the endogenous control. Assays were performed in all experimental conditions of the OVCAR3 cell line. Data are represented in the log10 scale using the sensitive experimental group as a calibrator. Bars represent the mean ± SE of two independent experiments performed in duplicate. **p ≤ 0.01 ***p ≤ 0.001. E Viability assays of the OVCAR3 cell lines transfected with pCMV6 (S-MOCK and R-MOCK) and with the overexpression vectors (R-PAX9 and R-FKBP1B). Each experimental group was exposed for 48 h to six different CDDP concentrations, and the data were normalized to each untreated control (set to 100%). The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each analyzed cell line. P-values < 0.01 indicated a significant change in drug sensitivity (Student’s t-test)