Increased DNA hypomethylation and growth inhibition by 5-Aza-CdR in G9a knockdown cells. (A) Cell growth curve and (B) DNA methylation levels at MAGE-A1 and p16exon2 locus, of G9a-knockdown (shG9a5) and control (NS) HCT116 cells treated with either PBS or 5-Aza-CdR. Cells were treated with the drug for 24 h. The drug containing media was then replaced with fresh media. The cells were kept in culture and cell counts and DNA methylation levels were assessed at respective time points. For 5-Aza-CdR treatment, samples examined were taken after 3 (Day 3) and 15 (Day 15) days of drug treatment. For PBS control treatment, sample examined was taken after 3 days of drug treatment. DNA methylation levels were measured using Ms-SNuPE assay as described in the Methods section. Data represent mean and range of methylation levels from two independent biological replicate drug treatment experiments, in each measuring methylation levels at three independent CpG sites within each locus.