A, Methylated or mock methylated promoter region (-1106/+45) was cloned into luciferase expressing vector pGL3-Basic and transiently transfected into A549 cells. SssI[ins] indicated that insert was densely methylated with SssI methylase (methylated mCG sites), HpaII[ins] indicate that insert was methylated with HpaII methylase (methylate CmCGG sites). B-D, effect of demethylating reagent 5-aza-dC on EC-SOD gene transcription. Cells were treated with 5-aza-dC at indicated concentrations for 4 days, RNA purified, reverse transcribed and amplified using primers specific for EC-SOD, TRAG3 (positive control for A549 and MRC5 cells) or GSTP1 (positive control for Hep3B cells) genes. GAPDH was used as normalization control. E, Exposure of A549 cells to TSA alone or in combination with 5-aza-dC was unable to further increase the EC-SOD transcription. A549 cells were exposed to 1.5 μM TSA and/or 1.0 μM 5-aza-dC for 4 days.