3t3 l1 mbx

The mouse pre-adipocyte, 3T3-L1 MBX (ATCC, Manassas, VA, USA) was cultured in growth medium (Dulbecco’s Modified Eagle’s Medium (Hyclone, UT, USA), high glucose, containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Welgene, Daegu, Republic of Korea) at 37 °C in a humidified atmosphere of 5% CO₂.
When the 3T3-L1 MBX cell density was confluence to differentiate into mature adipocytes, the culture medium was replaced with MDI media (growth medium supplemented including 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, ST. Louis, MO, USA), 0.5 µM dexamethasone (Sigma-Aldrich), and 5 µg/mL insulin (Sigma-Aldrich)). After MDI induction for 2 days, the differential media were exchanged for growth medium supplemented with 5 μg/mL insulin (insulin medium). The medium was changed every 2 days. After 4 days of exposure to the insulin medium, the media were replaced with growth media for another 2 days [28 (link),29 (link)].
The mature adipocytes were treated with or without pifithrin-α hydrobromide as a p53 inhibitor (PFT-α, 30umol/mL; MedChemExpress, Monmouth Junction, JN, USA) for 12 h [30 (link)], and then HIFU (Dot mode, 7MHz frequency, and 0.6 J energy) applied or not applied to the mature adipocytes and cultured for 2 days. Then, the samples were harvested.

The mouse fibroblast cell line 3T3-L1 MBX was purchased from ATCC^®. Cells were cultured in Advanced DMEM/F12 (Thermo Fisher Scientific Waltham, MA, USA; Ref. 12634010) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/ml streptomycin. Cells were grown and maintained in flasks and were not allowed to reach confluence. For individual experiments, cells were seeded onto 6-well or 96-well plates at low confluence. To induce adipocyte differentiation (using a protocol modified from [30 (link)]), on Day 0 cells were treated with 1 µg/mL insulin and 1 µg/mL dexamethasone (“Differentiation Medium”). After 48 h, the medium was switched to one containing 1 µg/ml insulin (“Maintenance Medium”), which was renewed every 2 days. Undifferentiated cells (“Day 0 Controls”) were processed for experiments directly on Day 0. All experiments were performed on non-differentiated and differentiated cells, corresponding to Day 0 and Day 7 of the experimental protocol, respectively. Treatment with GYY4137 was performed at D0 and D4, during the differentiation process.