Tamoxifen

All animal studies were performed according to protocols approved by the University Committee on Animal Resources at the University of Rochester Medical Center. Gclc^f/f mice were generated as described^{30 (link)} and crossed with the Rosa26-CreERT2 mouse strain (Jackson Labs, #008463) or Nrf2^f/f mouse strain (Jackson Labs, #025433). All animals were aged for at least 12 weeks before being used in their respective experiments. Tamoxifen (Sigma Aldrich, T5648) was administered by intraperitoneal injection at 160 mg/kg once daily for five days. For liver-specific genetic deletions, mice were injected via the tail vein with 2.5×10¹¹ GC of AAV-TBG-Cre (Addgene, 107787-AAV8) in PBS. For high-fat diet experiments, mice were fed a 60 kcal% fat high-fat diet (Research Diets, D12451i) following either Tamoxifen or AAV-TBG-Cre injection for 2–3 weeks. Control mice were fed normal rodent chow (LabDiet, 5053).

All animal experiments were performed according to an approved protocol (approval number: 2016-12-139, approved date: 9 August 2016), from the Institutional Animal Care and Use Committee of Asan Life Science Institute, Asan Medical Center, Seoul, Korea. Mice were housed in a temperature-controlled pathogen-free facility under a 12 h/12 h light/dark cycle (lights on at 08:00) with free access to water and a normal chow diet (Purina Rodent Chow, Seoul, Korea). To establish brown adipocyte-specific Atg7 conditional knockout mice, Atg7^fl/fl mice [20 (link),46 (link)] were crossed with UCP1-CreER^+/− mice [21 (link)]. Tamoxifen (1 mg/5 g body weight) (Sigma-Aldrich, St. Louis, MO, USA) dissolved in corn oil (Sigma-Aldrich) was orally administered to their off-spring for five consecutive days. To ablate Atg7 from newly generated brown adipocytes, every fifth week Tamoxifen was regularly administered to mice for five consecutive days [21 (link)]. In this experiment, Atg7^fl/fl mice and Atg7^fl/fl-UCP1-CreER^+/− mice (ATG7B KO) were used as the control and experimental groups, respectively, and their body weights were monitored every week for 1 year. For diet-induced obesity—1 week after Tamoxifen administration—the normal chow diet was switched to a 60% HFD (D12492, Research Diets, New Brunswick, NJ, USA).

Conditional activation of cre recombinase was induced by oral administration of tamoxifen (Sigma T5648). Per ml of in vivo oral delivery, tamoxifen was formulated as: 100 mg of tamoxifen dissolved in 100 μl 100% EtOH, followed by the addition of 1 ml of sunflower oil to achieve 100 mg ml⁻¹. The solution was sonicated in a water bath sonicator located at 4 °C for 10-s pulses. The solution was then placed in a water bath at 50 °C for 1 min until clear. Sonication and water bath steps were repeated until no particulates were noticeable. Animals were given 50 μl (5 mg per day) for three consecutive days by oral gavage. tamoxifen oral solution was aliquoted and stored at −20 °C and brought to administrable solution by 50 °C water bath for no longer than 5 min. The Tet-ON conditional system was activated with doxycycline chow (Research Diets, Inc. C11300–2000 with 2,000 p.p.m. doxycycline) 7 days post oral tamoxifen administration.