4 oht tamoxifen

Vegfr2^Y1212F/+ C57Bl/6: R26Stop^FLMYC allele mice and Vegfr2^Y1212F/+ C57Bl/6: Cdh5‐CreER mice were put together for timed breeding. At E9.5, 100 μl of 4OHT‐tamoxifen (Sigma, H7904) (10 mg/ml dissolved in peanut oil) was injected intraperitoneally into pregnant females. At E11.5, embryos were harvested and stored on ice in PBS until prepared for collagen embedding by dissecting away the head and the tail of each embryo. Genotyping of the embryos was done using the embryonic tailpiece. The embryos were embedded between two layers of rat tail type I collagen (Life technologies) and cultured in DMEM supplemented with 15% FCS and 30 ng/ml VEGFA for 10 days. Samples were fixed in 4% PFA and analyzed by microscopy after immunostaining with antibodies. Tile scan z‐stack images were taken with a Leica TCS SP8 Confocal Microscope with PMT and HyD detectors and LAS X Navigator software (Leica). Image analysis was done using ImageJ software (NIH) on a 2,000 × 2,000 μm region of the sprouting front of each explant.

Female nude mice, aged 5 to 6 weeks, were used for this study. 1 × 10⁷ cells were mixed with Matrigel (356234; BD Bioscience, Franklin Lakes, NJ, USA) at a ratio of 1:1 and the 100 µL cell mixture injected into the abdominal mammary fat pad of mice. When the tumors were palpable, mice were randomized into treatment and control groups where the treatment group received 0.5 mg of 4-OHT tamoxifen (H6278; Sigma, St. Louis, MO, USA) dissolved in ethanol and diluted in peanut oil (P2144; Sigma, St. Louis, MO, USA), and TCZ (1 mg/Kg and 2 mg/Kg) diluted in saline, given by subcutaneous injection. The mice were treated twice a week for 8 weeks.

Plasmid pBabepuro-myc-ER (#19128) was purchased from Addgene (Cambridge, MA, USA). U2OS, KMS27, and KMS28BM expressing MYC-ER (U2OS/MYC-ER, KMS27/MYC-ER, and KMS28BM/MYC-ER, respectively) were obtained through infection with the pBabepuro-myc-ER recombinant retroviral vector. Infected cells were selected using 1 µg/mL puromycin (Sigma-Aldrich, St Louis, MO, USA) and then cloned [17 (link),18 (link)]. U2OS/MYC-ER, KMS27/MYC-ER, and KMS28BM/MYC-ER were cultured with 1 µM 4OHT tamoxifen (Sigma-Aldrich, St Louis, MO, USA). Gene expression after 96 h (U2OS/MYC-ER), 72 h (KMS28BM/MYC-ER), or 6 h (KMS27/MYC-ER) of treatment was determined using real-time PCR.

MCF10A Src-ER cells were grown as described previously (8 (link)). 293T and U2OS cells were grown in DMEM (Life Technologies), supplemented with 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine (Life Technologies) at 37 °C in 5% CO₂. For SILAC labeling, MCF10A Src-ER cells were grown for 7 days in arginine- and lysine-free F-12/DMEM (Thermo Fisher) supplemented with stable isotope-labeled arginine (R0 or R10) and lysine (K0 or K8) (CKGAS), dialyzed horse serum (Dundee Cell Products), and the same supplements as normal cell culture. The cells were treated with a final concentration of 1 μm tamoxifen (4-OHT; Sigma), for 48 h and 0.3 μm 5-AzaC (Sigma), 24 h prior to 4-OHT treatment.