Fig. 4

Reversal of ZAR1 epigenetic inactivation in cancer cells by demethylation treatment. Various lung cancer cell lines as well as HEK and HeLa cells were treated with the demethylating agent 5-Aza-2’-deoxycytidine (0, 5 or 10 μM Aza). COBRA assay under Aza treatment shows demethylation of the ZAR1 promoter (a) and correlating reexpression of ZAR1 levels by semi-quantitative RT-PCR (b). PCR and digestion products were separated on 2% TBE agarose gel with 100 bp marker (m methylated, pm partially methylated, um unmethylated, pos. positive control, + TaqI-digested, − mock digested). c Real-time PCR quantification of ZAR1 reexpression after 5 μM Aza treatment, which was normalised to GAPDH. d Luciferase reporter assay was performed to determine the ZAR1 promoter activity. The artificial ZAR1 promoter was cloned into pRLnull and subsequently in vitro methylated. The promoters were transfected in HeLa cells and the ivm ZAR1 promoter showed full inactivation in contrast to its unmethylated construct and empty control construct. Student’s t test was used for statistical analysis