Animals were housed under standard conditions and maintained on a standard rodent diet

nol precipitation. The DNA recovered from each fraction was loaded onto a 1.5% low-melting-point agarose gel in a Tris acetateEDTA buffer, and the gel was cut to collect DNA ranging in size from 0.4 to 0.6 kb. A MinElute spin column was utilized to purify the DNA from the gel block. The 12 DNA preparations normalized with regard to size were quantified with a Quant-iT PicoGreen Kit and then divided into two merged preparations by the amount of the DNA in the upper and the lower halves of the fractions. A 250 pg sample of each PubMed ID: half was applied to quantitative PCR as described above to estimate the fold enrichment in the upper half relative to the lower half, which was plotted as a log2 value on the x-axis showing the CYP19 locus. Another SEVENS assay, in which six 1.6-ml fractions were collected, was also performed to analyze the fractional distribution of a given sequence. Following quantitative PCR for each fraction, the enrichment of a given sequence in each fraction relative to in whole merged fractions was calculated. PCR primers used in the assays are listed in S1 Chromatin immunoprecipitation assay Chromatin crosslinked in the two-step method was subjected to a ChIP assay as described previously. An aliquot containing 1 to 5 g of DNA ) was used for a single ChIP reaction. The primary antibodies were used as follows: anti-pan-histone H3, #ab1791, Abcam; anti-H3K27ac, #39685, Active Motif; anti-H3K27me3, #9733, Cell Signaling. Immunoprecipitation of protein-DNA complexes was performed with secondary antibody-conjugated Dynabeads M-280. To detect co-precipitated DNA, quantitative PCR was performed as described above. PCR primers used for these assays are listed in S1 Western blotting The cultured cells were suspended in western lysis buffer, 0.72 M 2-mercaptoethanol, 2% SDS, 0.005% bromophenol blue, and 8% glycerol) and boiled for 5 min. After centrifugation at 14,000 xg for 5 min, each supernatant was collected as a cell lysate. An EZQ Protein Quantitation Reagent was used to measure the protein concentration of each sample. Each protein sample was loaded onto a SDS-PAGE mini-gel; separated proteins were then blotted onto a polyvinylidene difluoride membrane. After sequential exposures to a primary antibody, a biotinylated secondary antibody, and a streptavidin-conjugated alkaline phosphatase, each membrane was developed with Western Blue. The primary antibodies were used as follows: anti-aromatase, #SM2222PS, Acris; anti–actin, 4 / 20 Chromatin Structures for Activity of the CYP19 Promoters #A1978, Sigma; anti-pan-histone H3, #ab1791, Abcam; anti-H3K27me3, #9733, Cell Signaling; anti-EZH2, #5246, Cell Signaling. Statistical analysis Statistical analysis was performed with Microsoft Excel. Each numeric data point was calculated from at least three independent measurements and is represented as mean SE. The p-values were generated using Student’s t-test. Results Assessment of transcription from three promoters in the human CYP19 gene The human CYP19 gene MedChemExpress ONX-0914 19697345″ title=View Abstract(s)”>PubMed ID: has multiple promoters; three of which are well-characterized. These three promoters–Ia, Ib, and Ic –are located in a region extending about 100 kb upstream of the protein-coding region. Liver carcinomaderived HepG2, granulosa cell carcinoma-derived KGN, and placental choriocarcinoma-derived JEG-3 cell lines each express CYP19 mRNA. First, we characterized the usage of each promoter in each cell line. Conventional RT-PCR with a separate forward primer for each unique first exon an