Fig. 3.

DUSP2 expression and methylation after 5-Aza-2′-deoxycytidine (Aza) treatment. a. Expression of DUSP2 in normal breast, kidney, liver, lung tissues (Agilent Technologies) and HEK293 cells was analyzed by qRT-PCR and normalized to ACTB (HEK293 = 1). b. A DUSP2 promoter fragment (454 bp) was cloned in the pRLnull vector and in vitro methylated (ivm). DUSP2 promoter constructs (DUSP2-pr.) were transfected in HEK293 cells and expression of renilla luciferase was measured and normalized to the expression of the co-transfected firefly plasmid pGL3.1 (pRLnull = 1). The analysis included the results (measurement in triplicates) of three independent experiments and significance is indicated (t-test) c. Expression analysis of DUSP2 in several cell lines after treatment with Aza (0, 5 and 10 μM) for four days. Expression of DUSP2 and ACTB was revealed by semi-quantitative RT-PCR and products (136 bp and 226 bp, respectively) were resolved on a 2 % agarose gel with a 100 bp marker ladder (M). d. Expression of DUSP2 in Aza treated H322 cells analyzed by qRT-PCR and normalized to ACTB. Data of three independent experiments, whereby each PCR was performed in triplicates and significance is indicated (t-test). e. Methylation analysis of DUSP2 after Aza treatment in H322 cells. Methylation of seven CpGs at proximal DUSP2 transcription start site (Seq2, see also Fig. 1) was analyzed by bisulfite pyrosequencing. Data were calculated from three independent experiments and significance is indicated (* = p < 0.01; 0 μM vs. 5 μM Aza)