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. 2023 Jun 22;72(11):1560–1573. doi: 10.2337/db22-1027

Figure 2.

Figure 2

Lipolysis activates FABP4 and AdEV secretion through a p53-dependent pathway. A: Differentiated 3T3-L1 adipocytes were treated with DMSO, 10 μmol/L Iso, 1 mmol/L Br-cAMP, or 15 μmol/L ASTAT for 4 h. p53 Activity was measured using the p53 transcriptional factor assay kit (catalog no. 600020, Cayman) and the results were normalized to the concentration of whole-cell lysate protein. B: Differentiated 3T3-L1 adipocytes were pretreated with DMSO or 15 μmol/L ASTAT for 2 h, followed by DMSO or 10 μmol/L Iso for 4 h. RNA was purified and p53 transcriptional targets expression were measured by qRT-PCR. C: Differentiated 3T3-L1 adipocytes were treated with DMSO, 10 μmol/L Iso, or 1 mmol/L Br-cAMP for 4 h. Total protein was harvested and acetyl-p53, phospho-p53, total p53, actin, and histone H3 were measured by immunoblot analyses by loading equivalent protein. D: Differentiated 3T3-L1 adipocytes were pretreated with DMSO or 20 μmol/L PFT for 2 h, then were treated with DMSO or 20 μmol/L FSK for an additional 2 h. Western blot analyses were used to measure intracellular NAMPT, FABP4, and actin levels, as well as secreted protein levels (NAMPT, FABP4, CD63, and TSG101) in the cell culture medium. E: Genetic knockdown of p53 (shp53) and control (shGFP) were made in 3T3-L1 cell lines by lentiviral transduction. The cells were treated with 20 μmol/L FSK for 4 h. Western blot analyses were used to measure intracellular p53, NAMPT, FABP4, and actin levels, as well as secreted protein levels (NAMPT, FABP4, CD63, and TSG101) in the cell culture medium. In all these experiments, the intracellular lysate was prepared from harvesting whole cells, and equal amounts of intracellular protein were loaded onto SDS-PAGE gels for immunoblots. For secreted samples, equal volumes of cell culture medium were used. For the AdEV markers CD63 and TSG101, AdEVs were pelleted from an equal volume of the cell culture medium, using a total AdEV isolation reagent (catalog no. 4478359, Invitrogen). The pellets were lysed and resuspended into an equal volume of RIPA buffer. F: Quantification of the protein secretion in p53 knockdown shown in D. All results represent the mean ± SEM. *P < 0.05, **P < 0.01.