Tamoxifen

Specific pathogen-free (SPF) C57BL/6 J mice and R26-tdTomato were purchased from the Jackson Laboratory. VE-cadherin-Cre-ER^{T2 28}, Mst1^{flox/flox27 (link)}, Mst2 null^{34 (link)}, and Foxo1^{flox/flox38 (link)} mice were transferred, established and bred in SPF animal facilities at KAIST and fed with free access to a standard diet (PMI Lab diet) and water. In order to induce Cre activity in the Cre-ER^T2 mice, tamoxifen (Sigma-Aldrich, T5648) was given with following dosages and schedules: for neonatal mice, 100 μg of tamoxifen dissolved in corn oil (Sigma-Aldrich, C8267) was injected into the stomach daily from P1 to P3; for OIR mice, 200 μg of tamoxifen was injected intraperitoneally (i.p.) daily from P12 to P14; for adult mice aged over 8 weeks, 2 mg of tamoxifen was injected i.p. for 5 consecutive days from the indicated time point. For anesthesia, mice were injected i.p. with the anesthetic solution (ketamine 40 mg/kg and xylazine 5 mg/kg). We complied with all ethical regulations for animal testing and research and performed all animal experiments under the approval from the Institute Animal Care and Use Committee (No. KA2017-31) of KAIST.

For neonatal tamoxifen (MilliporeSigma) administration, tamoxifen was dissolved in 100% corn oil (MilliporeSigma) at 10 mg of tamoxifen per mL. At P0, pups were injected once subcutaneously with 30 μL of 10 mg of tamoxifen per mL. For adult tamoxifen administration, tamoxifen was dissolved in a solution of 10% ethanol (200 proof, VWR) in corn oil at 20 mg of tamoxifen per mL. Adult 8-wk-old mice were intraperitoneally injected with 100 mg of tamoxifen per kg of body weight once per day for three consecutive days for single-cell suspension and immunohistochemistry. Adult ∼4-mo-old mice were injected with 100 mg of tamoxifen per kg of body weight once per day for five consecutive days for behavioral experiments. BrdU (MilliporeSigma) was dissolved in sterile normal (0.9%) saline at 5 mg of BrdU per mL. One dose of 50 mg of BrdU per kg of body weight was injected subcutaneously at P7.

Tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 mg/ml corn oil (Sigma-Aldrich, St. Louis, MO, USA) and 1 mg/20 g body weight per day of intraperitoneal Tamoxifen was given to 6-week-old mice for 5 consecutive days in order to induce Cre recombination. Control mice also received Tamoxifen at the same dose and frequency. Intraperitoneal injection of exendin-4 (Sigma-Aldrich, St. Louis, MO, USA) was performed 2 weeks after Tamoxifen injection. exendin-4 (0.1 μg/mg of body weight) was administered twice daily, while saline was administered to control mice.

To induce CRE activation during embryonic or postnatal development, mice were injected with tamoxifen (Sigma-Aldrich T5648) solubilized in ethanol and diluted in corn oil (Sigma-Aldrich C8267). For embryonic activation, pregnant females received a single intraperitoneal injection of tamoxifen (0.1 mg/g) mixed with progesterone (0.05 mg/g, Sigma-Aldrich, P3972) to counteract the estrogen agonist effects of tamoxifen. Cesarean sections were performed on pregnant females at E19.5 and pups were reared with foster mothers. For postnatal activation, pups received one intragastric injection of tamoxifen (50 μg) for three consecutive days starting from P1.

A stock solution of 30 mg tamoxifen (T5648, Sigma Aldrich, St. Louis, MO) in 1 ml of corn oil was prepared. To avoid premature abortion of fetuses due to tamoxifen, 0.2 mg β-estradiol (20 mg/ml of ethanol; E8875, Sigma Aldrich, St. Louis, MO) was added per ml of tamoxifen stock solution. On designated gestation days at noon, pregnant females were gavaged with the tamoxifen containing β-estradiol stock solution at 1 mg/10 g body weight. The morning of a found plug was considered as embryonic day 0.5.