β estradiol

S.pombe strains used in this study are listed in S1 Table. Yeast strains are constructed by either random spore method or by tetra analysis. Yeast cells are grown on YE+5S medium (add with 5 supplements including histidine, uracil, lysine, leucine and adenine) or YE+4S medium (add with histidine, uracil, lysine and leucine, except adenine is provided by yeast extract, it is sufficient to provide the growth for cells with ade6⁺ transgene inserted in the centromere or mating type locus and for colony color differentiation). Solid malt extract (ME) medium or synthetic medium without nitrogen (EMM-N) is used for mating and sporulation. β-estradiol (Sigma) at 100nM in YES is used for growth of psf1-HBD mutants, with β-estradiol at 0.1nM in YES for psf1-HBD depletion.

All drugs were maintained as stock solutions in DMSO, and stock solutions were stored at −80 and working stocks at −20 unless otherwise mentioned. 4-OHT (Sigma-Aldrich, cat# H7904) and fulvestrant (SelleckChem, cat# I4409) were purchased, and stocks were diluted to 10-mmol/L working stocks for all experiments other than dose curves, where specified concentrations were used. For all experiments, cells were treated 24 h after plating, and thereafter every 48 h until completion of experiment. For mouse xenograft experiments, fulvestrant concentrations of 250-mg/kg body weight were prepared in corn oil, freshly on day of injection and administered subcutaneously. Beta-estradiol was purchased from Sigma-Aldrich (cat# E8875), maintained in sterile, nuclease-free water, and diluted to obtain 10-mmol/L stocks for in vitro experiments. For mouse xenograft experiments, 17 β-estradiol was maintained in 200-proof ethanol at 2.7-mg/ml stock solution and added to drinking water twice a week at a final concentration of 8 μg/mL (cat# E2758; Sigma). For experiments involving Chloroquine (Selleckchem, cat#S4157), cells were treated at 50 μM for 16 h before end of assay. Lapatinib (SelleckChem, cat#S2111) and Neratinib were used at specified concentrations. Lapatinib tablets were used at 100 mg/kg in chow from Research Diets, Inc for tumor growth assays.

BALB⁄c-nude mice (16-22 g, female) were subcutaneously inoculated under the right shoulder with 5 ×10⁷ cells/flank. One week before injection, the mice transplanted with Cx43-deficient and its scramble T47D/TS cells were pretreated with drinking water containing 1 μM β-estradiol (Sigma-Aldrich). The mice transplanted with Cx43-overexpressed and control T47D/TR cells were provided with drinking water supplemented with 1 μM β-estradiol (Sigma-Aldrich) and pasted with estradiol sustained-release patch (1.25 mg per mice) (Zhejiang Yatai Pharmaceutical Co., Ltd., Zhejiang, China). When the volume of tumors reached to 100 mm³, the mice were intragastrically administrated with vehicle (control) or 30 mg/kg TAM for 10 days. Tumor dimensions were measured by a caliper every 2 days. The tumor volume (V) was calculated as (length×width²) × 0.5. The relative tumor volume (RTV) of each tumor was defined as the ratio of the volume at a given time and the volume prior to treatment 24 (link). On day 10, after their tumor sizes were measured, all mice were sacrificed and the xenografts were excised and weighted. Meanwhile, the expression of EMT biomarkers of xenografts were detected. All animal experiments were approved by the Guidance Suggestions for Caring for Laboratory Animals issued by the Ministry of Science and Technology of China in 2006.

To induce TIR1 using the β-estradiol system, cells, grown in leucine-deficient yeast minimal media (YMM) to OD₆₀₀ 0.7, were treated with 10 µM β-estradiol (Sigma-Aldrich #E8875; dissolved in 100% ethanol) to induce TIR1 expression and 0.75 mM Indole-3-acetic acid (IAA; auxin) (Acros organics #122160100) to deplete Spt5-AID*, for 40 min. To deplete Paf1-AID*, cells grown in YPDA medium to OD₆₀₀ 0.7 were treated with 0.75 mM IAA for 30 min. After incubation with auxin, samples were taken for protein, RNA and chromatin extraction as described below.

All strains expressing NUP-RITE constructs were grown to mid-log phase in SCD supplemented with 300 μg/mL hygromycin B (Roche) to select for non-recombined cells. Prior to imaging, cells were centrifugally collected and recovered for 1 hr in SCD without hygromycin B. Recombination was induced by addition of β-estradiol (1 µM f.c., Sigma-Aldrich) and cells were imaged 3 hr post induction.
Strains expressing NUP170-RITE constructs were grown to mid-log phase in SD-URA to select for non-recombined cells. Prior to imaging, recombination was induced by addition of β-estradiol (1 µM f.c., Sigma-Aldrich) and uracil and cells were imaged ~30 min (new Nup170-RITE) or ~5 hr (old Nup170-RITE) post induction.