17β estradiol

To express the silencing hairpin (Zct1hp or GFPhp), transgenic C. roseus hairy roots were induced with 5μM 17β-estradiol (Fisher Scientific) on day 26 (late-exponential growth phase). Previously, induction with 5μM 17β-estradiol showed strong and tightly regulated expression of GFP in control transgenic C. roseus hairy roots [31 ]. Stock solutions (5mM) of 17β-estradiol were prepared in dimethyl sulfoxide (DMSO, Sigma Aldrich), and 50μL of stock solution was added to 50mL of culture media to achieve a final concentration of 5μM 17β-estradiol. Uninduced cultured were treated with 50μL of DMSO.
Cultures were elicited with MJ (≥ 95%, Sigma Aldrich) 24 h after induction with 17β-estradiol. Stock solutions were prepared in ethanol (200 proof, ACS/USP grade, Pharmco-AAPER) and added to 50mL of culture media to achieve final concentrations of 250μM or 1000μM MJ. Cultures were harvested 8, 24, and 48 h after MJ addition for mRNA analysis or after 3, 5, and 7 days for TIA metabolite analysis. C. roseus hairy roots were blotted and flash-frozen in LN₂.

Tumor pieces of 30–60 mm³ obtained from patient primary tumors or metastatic lesions at time of biopsy were immediately implanted into the mammary fat pad (surgery samples) or the lower flank (metastatic samples) of 6-week-old female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice (M. musculus, Charles Rives). Mice were housed in air-filtered flow cabinets with a 12-hours light cycle at 18–23 °C, 40–60% of humidity and food and water ad libitum. Mice were continuously supplemented with 1 µmol/L 17β-estradiol (Sigma-Aldrich) in their drinking water, an amount shown to be sufficient to reach serum levels and uterine growth in ovariectomized female mice similar to the ones obtained with other mechanism of 17β-estradiol supplementation. Upon growth of the engrafted tumors, a tumor piece was implanted into the lower flanks of new recipient mice for the model perpetuation. In each passage, flash-frozen and formalin-fixed paraffin embedded (FFPE) samples were taken for genotyping and histological studies. STG201 was generated in CRUK/UCAM as previously reported^{21 (link)} and PDXs BB3RC31, BB6RC39, BB6RC87, and BB6RC160 were generated in Manchester Breast Center. Both laboratories are members of the EuroPDX consortium.

Commercially available human male and female iPSC-derived cardiac myocytes (iPS-CMs, Axol Bioscience, UK) were prepared according to the manufacturer’s instructions. Briefly, cardiac myocytes (1 ml, stored frozen in liquid N₂) thawed in a 37 °C water bath were initially suspended in 1 ml Axol Complete Cardiomyocyte Medium (warmed to 37 °C). An additional 8 ml of medium was added and mixed by gentle inversion. Cardiac myocytes were seeded (25,000 cells/well) in 1.5 ml medium in 12 well culture plates that contained glass coverslips (15-mm diameter, Warner Instruments, CT) pre-coated with Matrigel™ (Corning Life Sciences, MA). Cells were incubated for 24 h at 37 °C, 7% CO₂. Non-adherent cells were removed by rinsing with media. After 7 days in culture, 17-β-estradiol (Sigma) was added to the media at a final concentration of 1 nM. The iPS-CMs were kept in 17-β-estradiol for 24–48 h before measuring I_Ca,L or I_NCX by voltage-clamp technique. Control cells were treated with media containing DMSO (< 0.1%). Cultured iPS-CMs had a tendency to mature and exhibit spontaneous contractions by the 7th day of incubation. The male and female iPS-CMs used in this study had common features found in all iPS-CMs: spontaneous automaticity and contractions and stable ionic currents for weeks, as previously reported [34 (link)].