Sc 2028

WM852 cells were transfected with the expression plasmid NICD3-pCLE (11 (link)) and 48 hours later, the chromatin was processed by the SimpleChIP kit according to the manufacturer’s instructions (Cell Signaling Technology). The chromatin was precipitated using antibodies against Notch3 (M-134, Santa Cruz Biotechnology or 8G5, Cell Signaling Technology) or normal goat or rat IgG antibody (sc-2028 or sc-2026, Santa Cruz Biotechnology). DNA was purified using the PCR Purification kit (Macherey-Nagel) according to the manufacturer’s instructions.
After DNA purification, the WNT5B promoter regions were amplified and analyzed by qPCR (Supplemental Table 4).

MG tumor cell pellets (at least 3 × 10⁶ cells) or Gfi1-infected NIH-3T3 cells (ATCC CRL-1658) were resuspended in lysis buffer (150 mM NaCl, 20 mM Tris, 1% Triton X-100) with protease inhibitors (Roche, cat #1836153). Lysates were precleared for 30 min with Protein G agarose beads (Millipore #16-201D, Cell Signaling #37478). Totally, 10% of the sample was saved as input, and the rest was split in half for immunoprecipitation with experimental and control antibodies. Samples were incubated with antibodies specific for Gfi1 (1 µg, Santa Cruz sc-8558), Lsd1 (1 µg, Abcam ab17721) or CoREST (1 µg, Millipore cat #07-455), or with isotype control antibodies (1 µg, Santa Cruz sc-2028) for 1 h. Protein G beads were added to the samples and washed three times before preparing for Western blot by adding 4× sodium dodecyl sulfate (SDS) sample buffer and boiling.

The Ch-IP protocol is based on the protocol of Bryan Dynlacht and Richard Young laboratories. For the Sox17—CCNE1 experiment, HLMVECs transduced with pMXs-ms-Sox17 (a gift from S. Yamanaka^{79 (link)}, Addgene plasmid #50781) were crosslinked by using 1% formaldehyde (Fisher Scientific #F79–1), washed three times with cold PBS, and resuspended in cell lysis buffer. The nuclei portion was resuspended in nuclear lysis buffer and sonicated to break down the genomic DNA using S220 Focused-ultrasonicator (Covaris). After centrifugation, the supernatant was immunoprecipitated with 5 μg anti-Sox17 (R&D #AF1924) or an equal amount of goat IgG (Santa Cruz #sc-2028). The DNA obtained from the IP was amplified by qPCR with primers specifically recognizing different binding sites in CCNE1 promoters (Primer information included in Supplementary Table 2). For the HIF-1α-SOX17 experiment, HLMVECs cultured in normoxic or 1% O₂ hypoxic conditions were processed as above and then immunoprecipitated with 5 μg anti-HIF1α (Santa Cruz #sc-10790) or an equal amount of rabbit IgG (Santa Cruz #sc-2027). The DNA obtained from the IP was amplified by qPCR with primers specifically recognizing different HRE sites in SOX17 promoters (Primer information included in Supplementary Table 2).

Estrogen-deprived MCF7 cells treated with either 10 nM estradiol (E2), 2.5 µM Senexin A or a combination of E2 and Senexin A for 12hr and ChIP assays were performed as previously described [38 (link)]. Briefly, cells were fixed with 1% (v/v) formaldehyde, harvested for whole cell lysate preparation and immunoprecipitation was performed with 2 µg of antibodies against CDK8 (sc-1521, SantaCruz), ER-α (sc-543, SantaCruz), RNA-Pol II-S2P (C152000005, Diagenode) and rabbit and goat IgG (sc-2027, sc-2028, Santa Cruz) and enriched DNA was analyzed by q-PCR with ChIP primers (Supplemental Table 2).