Figure 4

KCTD11 is silenced by methylation in HCT-15 and HCT-116 cell lines. A CpG island, containing 72 CpG dinucleotides (indicated by the vertical lines) was identified from -522 bp to the +70 bp of KCTD11 promoter http://www.ebi.ac.uk/emboss/cpgplot/. (B-C) HCT-15 and HCT-116 colorectal carcinoma cells were cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine (Sigma), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma), and treated with 2 μM of Aza (Sigma) daily for 1 and 3 days, respectively. RNA was extracted by using Rneasy Mini Kit (Qiagen) and cDNA was generated from 1 μg of DNase I treated RNA using MuLV Reverse Trascriptase (Applied Biosystems). (B) KCTD11 mRNA level was analyzed by q-RT-PCR, normalized to GAPDH. The demethylating agent, Aza increased KCTD11 expression. Primers sequences are available in additional file 2. (* indicates p < 0.005; ** indicates p < 0.001). All measurements were performed in triplicates. Values are the means ± S.D. (C) Representation of methylated CpG found in KCTD11 promoter after bisulfite treatment of HCT-15 and HCT-116 cell lines. Closed and open boxes indicate methylated and unmethylated CpG sites, respectively. Horizontal lanes indicate methylation status of several PCR-amplified sodium bisulfite-treated HCT-15 and HCT-116 cDNA clones. PCRs were performed using two primer sets amplifying the CpG dinucleotides from 1 to 30 and from 31 to 72 [additional file 2; arrows on Fig. 4A]. PCR products were cloned into pCRII-TOPO-TA vectors (Invitrogen) and colonies screened by blue/white selection. Plasmid DNAs from at least ten colonies were sequenced and analyzed for C-T conversion.