4 hydroxytamoxifen

Conditional LoxP-flanked Drosha^fl/fl Gt(ROSA)26Sor^CreERT2 ESCs have been described previously ^{28 (link)}. All ESCs were cultured in KnockOut DMEM, supplemented with 10% heat-inactivated fetal bovine serum (GE Healthcare Life Sciences), 5% KnockOut Serum Replacement (Gibco), 1% sodium pyruvate (Gibco), 1% non-essential amino acids, 1% penicillin–streptomycin-glutamine (Gibco), 0.1 mM 2-mercaptoethanol and 10³ U/mL LIF on a Mitomycin C-inactivated mouse embryonic fibroblasts.
Deletion of the floxed Drosha allele was achieved by adding 100 nM 4-hydroxytamoxifen (Sigma-Aldrich) to the Drosha^fl/fl Gt(ROSA)26Sor^CreERT2 ESCs for 72 h. The medium was then replaced (without 4-hydroxytamoxifen) for a further 48 h to allow for depletion of Drosha-dependent miRNAs before analysis.
Deletion of the floxed Dicer allele was achieved by transducing the Dicer^fl/fl ESCs with a Cre-expressing retrovirus^{53 (link)}. The virus also contained a GFP reporter that allowed for the sorting of transduced cells. GFP⁺ ESCs were sorted 3 days after transduction, then cultured for a further 2 days before analysis.

For the growth rate measurement, 5 × 10⁴ MDCK-5KO-EKARrEV-NLS-loxP-NRG1 or MDCK-5KO-EKARrEV-NLS-loxP-NRG1-CreERT2 or 2.5 × 10⁴ MDCK-5KO-EKARrEV-NLS-loxP-NRG1-CreERT2 and 2.5 × 10⁴ MDCK-WT cells were seeded in a six-well plate (No. 140675; Thermo Fisher Scientific) and cultured in DMEM containing 10% FBS. After 24 h incubation, 1 μM 4-hydroxytamoxifen (no. 579002; Sigma-Aldrich) or DMSO (no. 13445-74; Nacalai Tesque) or 1 μM 4-hydroxytamoxifen and 10 ng/ml EGF (no. E9644; Sigma-Aldrich) was added. Fluorescence images of fixed locations were acquired with a UPlanFL-PH 10x/0.3 objective lens (Olympus) every 24 h. Images were binarized to count the number of nuclei by FIJI.

The construction of PUER-derived reporter cell lines is described elsewhere [33 (link)]. PUER cells were cultured and differentiated as described previously [12 (link), 34 (link)]. Briefly, PUER cells were grown in complete Iscove’s Modified Dulbecco’s Glutamax medium (IMDM; Gibco, 12440061) supplemented with 10% FBS, 50μM β-mercaptoethanol, 5ng/ml IL3 (Peprotech, 213–13). PUER cells were differentiated into macrophages by adding 200nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904–5MG). Cells were differentiated into neutrophils by replacing IL3 with 10ng/ml Granulocyte Colony Stimulating Factor (GCSF; Peprotech, 300–23) and inducing with 100nM 4-hydroxy-tamoxifen (OHT; Sigma, H7904–5MG) after 48 hours.

Human ER-positive breast cancer cell lines MCF7 and T47D (ATCC), and HEK293T (ATCC) cells were cultured in DMEM (Hyclone) with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Hyclone) at 37 °C in a humidified 5% CO₂ atmosphere. Tamoxifen-resistant (TamR) MCF7 and T47D cells were generated as described previously [63 (link)]. In brief, cells were cultured in the presence of increasing concentrations of 4-hydroxytamoxifen (Sigma-Aldrich) starting at 0.5 μM, and finally gradually increased up to 5 μM 4-hydroxytamoxifen when the growth of the cells could not be inhibited in this concentration. In parallel, parental MCF7 and T47D cells were cultured under identical conditions without tamoxifen. SP600125, etomoxir and puromycin was obtained from MedChem Express.

The MCF-7^TamR cell line was generated as previously described [41 (link)]. Briefly, MCF-7 cells were grown continuously in medium containing 1 μM 4-hydroxytamoxifen (Sigma-Aldrich) for 3 months and 3 μM 4-hydroxytamoxifen for at least 9 months.

For genetic cell labelling we crossed tamoxifen-inducible Csf1r^MCM, Tie2^MCM, and Kit^MCM with Rosa26^eYFP, Rosa26^GFP, or Rosa^mTmG reporter mice. In Csf1r^MCM embryos recombination was induced by single injection at E8.5 of 75 µg per g (body weight) of 4-hydroxytamoxifen (Sigma) into pregnant females. The 4-hydroxytamoxifen was supplemented with 37.5 µg per g (body weight) progesterone (Sigma) to counteract the mixed oestrogen agonist effects of tamoxifen, which can result in fetal abortions. In Tie2^MCMRosa26^eYFP embryos recombination was induced by treatment of pregnant females by gavage at different timepoints at E7.5 and E10.5 with a single dose of 2.5 mg tamoxifen (Sigma) and 1.25 mg progesterone. In Kit^MCM animals recombination was induced in 6–8-week-old adult animals using oral tamoxifen feeding (400 mg/kg diet, which approximates to a daily dose of 40 mg/kg mouse body weight; purchased from Envigo).