Ab23738

Paraffin embedded prostate cancer tissue blocks were from the Vancouver Prostate Centre Tissue Bank. Patient consent was reviewed and approved by the University of British Columbia Clinical Research Ethics Board (certificate no. H09-01628). Immunohistochemical staining was conducted as previously described ^{56 (link)} using the Ventana DiscoverXT Autostainer (Ventana Medical System) with enzyme labeled biotin streptavidin system and solvent-resistant DAB Map kit. Antibodies used in IHC include anti-FoxA1 (Abcam; ab23738), anti-SYP (Abcam; ab32127), anti-chromogranin A (Millipore; MAB5268), and anti-IL-8 (R&D systems).

ChIP experiments were conducted as described previously (16 (link)) and were done in duplicates. MCF7, T47D, MDAMB134 and SUM44 cells were cultured for three days in HD conditions then treated with 10nM estradiol for 45 minutes. For the ChIP experiments with tamoxifen we used 10nM 4-hydroxytamoxifen for 45 minutes. Chromatin from 20 million formaldehyde-fixed cells was sonicated to a size range of 200–300 bp. Solubilized chromatin was immunoprecipitated with a mix of the ER antibodies Ab10 (Thermo Fisher Scientific) and SC-543 (Santa Cruz), a mix of the FOXA1 antibodies ab5089 and ab23738 (Abcam), GATA3 antibody D13C9 (Cell signaling) or H3K27Ac antibody (C15410196, Diagenode). The same antibodies were used in each experiment for all the cell lines. The samples were reversed crosslinked, treated with proteinase K, and DNA was extracted. Libraries were sequenced using 75 bp paired-end reads on the Illumina Nextseq500 at the Dana-Farber Cancer Institute.

Using a 2-mm² core needle, approximately three cores (TF) were extracted from areas circled on each slide. Frozen cores were pulverized using the Covaris CryoPrep system. Tissue was then fixed using 1% formaldehyde with methanol for 18 min at room temperature, and fixation was quenched with 2M glycine. Chromatin was sheared to 300–500 bp in size using the Covaris E220 ultrasonicator. The resulting chromatin was incubated overnight with 6 µg of antibody—to AR (E2724, Spring Biosciences), HOXB13 (sc-66923, Santa Cruz Biotechnology) or FOXA1 (ab23738, Abcam)—bound to protein A and protein G beads (Life Technologies). A fraction of the sample was not exposed to antibody and was used as control (input). Samples underwent cross-linking reversal and were treated with RNase and proteinase K, and DNA was extracted. Concentrations of ChIP DNA were quantified by Qubit fluorometer (Life Technologies). DNA sequencing libraries were prepared using the ThruPLEX-FD Prep kit (Rubicon Genomics). Libraries were sequenced using 75-bp reads on the Illumina platform at the Dana-Farber Cancer Institute.