GoKawiilCell culture
MCF7 (HTB-22), ZR-75-1 (CRL-1500), CAMA-1 (HTB-21), MDA-MB-453 (HTB-131), MDA-MB-231 (HTB-26), OVCAR-3 (HTB-161) and Kuramochi (JCRB0098) cells were obtained from the American Type Culture Collection. MCF7 ESR1Y537S cells were provided by S. Chandarlaparty35. MCF7, ZR-75-1, MDA-MB-231 and CAMA-1 cells were cultured in RPMI-1640 medium (11875-093, Gibco) supplemented with 10% fetal bovine serum (FBS; SH30054.03, GE HyClone; SFBS-AU, Bovogen Biologicals), 1× GlutaMAX (35050061, Gibco) and 1% HEPES (15630080, Gibco). OVCAR-3 and Kuramochi cells were cultured in RPMI-1640, supplemented with 10% FBS and 1× GlutaMAX. MDA-MB-453 cells were cultured in DMEM supplemented with 10% FBS. All cells were maintained at 37 °C and 5% CO 2 in a humidified incubator. Cells were maintained below 90% confluency and routinely tested to confirm lack of mycoplasma contamination via PCR testing. Cell lines were authenticated by STR fingerprinting.
In vitro drug treatment studies
Abemaciclib methanesulfonate (HY-16297) and palbociclib isethionate (HY-A0065) were purchased from MedChemExpress. INX-315 was obtained from Incyclix Bio. Fulvestrant (S1191) and imlunestrant (E1301) were purchased from Selleck Chemicals. All drugs were diluted in dimethyl sulfoxide (DMSO) for in vitro studies. Unless otherwise noted, abemaciclib and palbociclib were used at 500 nM, INX-315 was used at 300 nM, fulvestrant was used at 5 nM and imlunestrant was used at 40 nM.
For cell cycle analysis by BrdU, MCF7, OVCAR-3 and Kuramochi cells were treated with DMSO or drug (abemaciclib, palbociclib or INX-315, respectively) for 2 days; ZR-75-1 cells were treated for 4 days (abemaciclib or palbociclib). For CUT&RUN, MCF7, OVCAR-3 and Kuramochi cells were treated for 2 days, and ZR-75-1 cells were treated for 4 days. For dose matrix drug combination assays, cells were treated with abemaciclib and fulvestrant in a dose matrix using the Tecan D300e Digital Dispenser, with a treatment length of 5 days.
For hormone-deprivation and stimulation experiments, cells were cultured in phenol-free RPMI-1640 (11835030, Gibco) supplemented with 1× GlutaMAX, 1% HEPES and 10% charcoal-stripped FBS. Oestradiol (E2758, Sigma) dissolved in ethanol was used at 1 nM.
Cell cycle analysis by flow cytometry
Thirty minutes before end point, 10 μM BrdU (B5002, Sigma) was applied to the cells. Cells were stained using the Near-IR Fixable Viability Stain (L34976, Invitrogen) for 20 min at room temperature. Cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (00-5523-00, eBioscience). DNA was denatured using 2 N HCl + 0.5% (v/v) Triton X-100 for 30 min. The acid was neutralized using 0.1 mol l−1 Na 2 B 4 O 7 •10H 2 O (pH 8.5) followed by 0.5% BSA in PBS. Cells were then stained with BrdU antibody (clone 3D4, 560209, BioLegend; 1:20) for 1 h. Before acquisition on the BD A3 Symphony, DNA was stained using FxCycle Violet Stain (F10347, Invitrogen). A minimum of 10,000 live cell events were recorded for each sample and data were analysed using FlowJo (v10.8.1).
Dose matrix drug combination assays
Cell counts
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