For thorough sequencing of the methylated sites, the bisulfite treated DNA was subjected to PCR to amplify the region. The primer sequences used were listed in Additional file 1 Figure S1. The PCR conditions were 94 C for 2 min, followed by 30 cycles of 94 C for 20 s, 55 C for 20 s and 72 C for 30 s, with a final extension at 72 C for 5 min. The resulting products were purified using a Qiaex II gel ex traction kit and then subjected to direct sequen cing in both direction. The methylation ratio of each CpG site for each tissue was calculated as the percentage of methylation versus the methylated plus unmethylated sites. Quantitative real time reverse transcription PCR analysis MTO1 and MRPL41 expression levels were measured by quantitative real time RT PCR analysis using cDNA syn thesized from 5 ug of total RNA and a reverse transcrip tion kit.

One microliter of cDNA was used for the PCR, and duplicate reactions were per formed for each sample using a Kapa SYBR Fast qPCR Kit with Inhibitors,Modulators,Libraries gene specific primers on an ABI 7500 instrument. The primers used for these selected genes are listed in Additional file 1 Figure S1. RNA quantity was normalized to GAPDH content, and gene expression was quantified according to the method. Chromatin immunoprecipitation PCR ChIP assays were performed Inhibitors,Modulators,Libraries using an EZ ChIP Chromatin Immunoprecipitation kit as described in the suppliers protocol. Briefly, the cross linked chromatin was sonicated after cell lysis and then incubated with antibodies against ER at 4 C overnight.

The immunocomplex was precipitated with Protein A agarose, and Inhibitors,Modulators,Libraries the beads were washed, sequentially treated with 10 ul of RNase A and 75 ul of Proteinase K, and incubated at 65 C overnight to reverse cross link the chromatin. The DNA was recovered by phenol chloroform extraction and coprecipitation with glycogen, and dissolved in 50 ul of Tris EDTA buffer. DNA associated with the ER was amplified by PCR using 1 ul of the precipitated DNA. PCR primers were designed to amplify the ER responsive elements at the promoter. The PCR conditions were 30 cycles at 94 C for 40 s, Inhibitors,Modulators,Libraries 57 C for 1 min, and 72 C for 40 s. Luciferase assay The upstream region of MTO1 and MRPL41 was ampli fied by PCR from human chromosomal DNA and cloned into the MluI and HindIII sites of pGL2Basic luciferase vector. The PCR was performed using primers with 35 cycles at 94 C for 30 seconds, 55 C for 1 minute, then 72 C for 2 minutes.

100 ng of the recombinant luciferase expression vector was transiently transfected into 1 104 cells in 96 well culture plates using a transfection kit. Luciferase activity Inhibitors,Modulators,Libraries was measured 36 hours after transfection in three inde pendent cultures using a dual luciferase reporter assay system kit on a Molecular Devices Filter Max F3. The activity from the promoter spanning learn more R0 R4 of MTO1 and R0 R6 of MRPL41 was normalized with that from the promoter containing only R0 fragment of each gene.