Fig. 2

DNA methylation-associated silencing of selected genes comparing the trastuzumab-resistant and trastuzumab-sensitive cell line. a DNA methylation levels of TGFBI, CXCL2, and SLC38A1 in SK and SKTR cell lines by bisulfite pyrosequencing and b methylation-specific polymerase chain reaction (MSP) analysis. c Expression levels of TGFBI, CXCL2, and SLC38A1 in the unmethylated (SK) and methylated (SKTR) cell lines determined by qRT-PCR. d Restored expression of selected genes (TGFBI, CXCL2, and SLC38A1) after DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) in the SKTR methylated cell line by qRT-PCR. e Protein expression of all selected genes (TGFBI, CXCL2, and SLC38A1) in SK and SKTR cells before and after 5-aza-dC treatment by Western blot. f Analysis in AU and AUTR cell lines of TGFBI methylation by MSP (top) and (middle) its transcriptional (qRT-PCR) and (bottom) protein levels (Western blot) before and after 5-aza-dC treatment. In MSP, the presence of visible polymerase chain reaction products in lanes marked U indicates unmethylated sequences; the presence of products in lanes marked M indicates methylated sequences. In vitro methylated DNA (IVD) was used as a positive control for methylated sequences. DNA from normal lymphocytes (NL) was used as a negative control for methylated sequences. Results shown are representative of those obtained from three independent experiments, and b-actin was used as a control. Values from pyrosequencing and qRT-PCR were determined from triplicates and are expressed as the mean ± SEM. Significance of Mann-Whitney U test, **p < 0.01; *p < 0.05