Antibiotic antimycotic solution

Ovary and cervix-free uterine horns were washed with Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, CA) supplemented with 2% (v/v) antibiotic-antimycotic solution (Welgene Inc., Daegu, Korea). Subsequently, the washed uterine horns were split longitudinally, fragmented into fine pieces by surgical scissors, and digested by incubation in HBSS containing 1.5 mg/mL collagenase (Sigma-Aldrich, St. Louis, MO) for 45 min at 37°C. The digested cells were filtered through 100 μm nylon strainer (SPL, Pocheon, Korea) and washed with Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12; Invitrogen) containing 10% (v/v) heat inactivated fetal bovine serum (FBS; Welgene) and 1% (v/v) antibiotic-antimycotic solution (herein referred to as DMEM/F12 culture medium). A sedimentation step collected cell clumps in the tube bottom after separating the filtrated cells under unit gravity by incubating in a 15 mL tube at room temperature for 15 min. This procedure was repeated three times to eliminate debris included in cell clump. Subsequently, an adherent step separated attached ES and suspended EE cells by incubating the cells clump on a 100 mm culture plate at 37°C for 10 min that was repeated twice. Then two types of cells were isolated and enumerated using a hemocytometer, respectively.

3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (high glucose), which contained 10% FBS and 1× antibiotic–antimycotic solution (WelGENE Inc., Daegu, Republic of Korea). The cells were incubated at 37 °C in the presence of 5% CO₂. The viability of cells was determined using the Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim, Germany). Cells at a concentration of 4 × 10⁴ cells/well in 500 μl culture medium were seeded in 24-well plate. Incubated cells for 6 h at 37 °C and 5% CO2 and washed it with serum-free medium, then added 500 μl serum-free medium containing the extract or piperine. After 24 h, added 20 μl/well Cell Proliferation Reagent WST-1 and incubate the cells for 1 h. Shake thoroughly for 1 min on a shaker and measure the absorbance of the samples against a background control as blank using the microplate reader at 440 nm and 690 nm.

Specific antibodies were purchased from the following commercial sources: anti-Flag, anti-Myc, and anti-β-Actin from Sigma-Aldrich (USA); anti-Xpress (Xp) from Invitrogen (USA); and anti-GST, anti-TDAG51, and anti-PPARγ antibodies from Santa Cruz Biotechnology (USA). Murine preadipocyte 3T3-L1 and human embryonic kidney 293T and PlatE cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Korea) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1× antibiotic/antimycotic solution (Welgene).

LNCaP and 293T cells were purchased from the Korean Cell Line Bank (KCLB; no. 21573), Korean Cell Line Research Foundation, Seoul, Korea, in 2017. The cells were maintained in RPMI-1640 (Welgene, Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 0.1% antibiotic/antimycotic solution (Welgene) at 37°C in a 5% CO₂ humidified chamber.