Pft α

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.

IL-7 was purchased from Peprotech (Peprotech, Rocky Hill, NJ). P53 (1:1000, Proteintech Group, Chicago, IL, USA), AMPK-α (1:1000, Wanlei Bio, Shenyang, China), β-actin (1:1000, Proteintech Group,Chicago, IL, USA), GAPDH (1:1000, Proteintech Group,Chicago,IL, USA), anti-mouse IgG (1:2000, Proteintech Group, Chicago, IL, USA) and anti-rabbit IgG (1:2000, Proteintech Group, Chicago, IL, USA), mTOR (1:1000, Cell Signaling Technology, Beverly, MA), phosphorylated(p)-mTOR (1:1000, Cell Signaling Technology, Beverly, MA) and p-AMPK (1:1000, Cell Signaling Technology, Beverly, MA), LC3B (1:1000,Cell Signaling Technology, Beverly, MA), anti-IL-7 receptor antibody (A-IL-7R) (Santa Cruz, CA), P53 inhibitor: PFT-α (MedChemExpress, Monmouth, NJ, USA), AMPK inhibitor: Compound C (MedChemExpress, Monmouth, NJ, USA) and mTOR inhibitor: Rapamycin (MedChemExpress, Monmouth, NJ, USA).

p38 inhibitor SB203580 (10 μM), c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10 μM), extracellular signal-regulated kinase (ERK) inhibitor PD98059 (5 μM), PKA inhibitor H-89 (5 μM), c-Jun inhibitor SR11302 (5 μM), CREB inhibitor EML-425 (5 μM), and p53 inhibitor PFT-α (10 μM) are all purchased from MedChemExpress (Shanghai, China). All inhibitors were configured with dimethyl sulfoxide (DMSO; Solarbio, Beijing, China). Antibodies against Bax, Bcl-2, caspase 3, caspase 8, caspase 9, FasL, Fas, p53, his, phospho-p53(ser15), phospho-p53(ser46), and phospho-p53(ser20) were purchased from Cell Signaling Technology (Danvers, MA, United States). Monoclonal antibodies against CCN1, c-Jun, phospho-c-Jun, c-Fos, phospho-c-Fos, CREB, and phospho-CREB were obtained from ABclonal (Wuhan, China). Antibodies against PEDV (CH/SXYL/2016) N protein were stored in our laboratory (Xu et al., 2019 (link)).