C15100066

The ChIP assay was performed according to the protocol from the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) as described in detail in Vydra et al., 2019 (link). For each IP reaction, 30 µg of chromatin and 4 μl of mouse anti-ERalpha monoclonal antibody (C15100066, Diagenode) was used. For negative controls, chromatin samples were processed without antibody (mock-IP). Obtained DNA fragments were used for global profiling of chromatin-binding sites or gene-specific ChIP-qPCR analysis using specific primers covering the known EREs. The set of delta-Cq replicates (difference of Cq values for each ChIP-ed sample and corresponding input DNA) for control and test sample were used for ERα-binding calculation (as a percent of input DNA) and estimation of the p-values. ERE motifs in individual peaks were identified using MAST software from the MEME Suite package (v. 5.1.1) (Bailey et al., 2015 (link)). The sequences of used primers are presented in Supplementary file 7.

Cells were plated onto Nunc Lab-Tek II chambered coverglass (#155383, Nalge Nunc International, Rochester, NY) and fixed for 15 min with 4% PFA solution in PBS, washed, treated with 0.1% Triton-X100 in PBS for 5 min, and washed again in PBS (3 × 5 min). IF imaging was performed using primary antibodies: anti-HSP90 (1:200; ADI-SPA-836, Enzo Life Science), anti-HSF1 (1:300; ADI-SPA-901, Enzo Life Sciences), or anti-ESR1 (1:200; C15100066, Diagenode) and secondary Alexa Fluor (488 or 594) conjugated antibodies (Abcam). Finally, the DNA was stained with DAPI. Images were taken using Carl Zeiss LSM 710 confocal microscope with ZEN navigation software.

The MCF-7 cells were treated with either DMSO or 27-HC (1 µM) for 72 h, and the cells were lysed in Radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 6.8), 0.1% sodium dodecyl sulfate (SDS), 0.5% NP40, 150 mM NaCl, 0.5% sodium deoxycholate]. The cell lysate was subjected to immunoprecipitation (IP) by incubating with either 1 µg of ChIP-grade ERα antibody (Diagenode C15100066-100) or 1 µg control mouse IgG (CST 5415s) antibody overnight at 4°C with gentle rotation. The protein A dynabeads (Invitrogen 10001D) were incubated with 3% bovine serum albumin (BSA) for 45 min in the rotator at 4°C. The IP samples were further incubated with the beads for 2 h and washed with the lysis buffer thrice. The beads were reconstituted in 1× PBS and denatured in 4× sample SDS-PAGE gel loading buffer heating at 95°C. The beads were carefully removed, and the supernatant samples were separated on 12% SDS-PAGE. The separated proteins were further transferred to polyvinylidene difluoride (PVDF) membrane and blocked with 5% BSA for an hour and washed with Tris Buffered Saline with Tween (TBST). The blot was incubated with primary antibody (anti-Dnmt3b #Ab2851, ERα antibody Diagenode C15100066) overnight at 4°C. The blots were further incubated with suitable secondary antibody for 90 min, and the membrane was developed using ECL reagent (Bio-Rad).