Figure 7.

mTOR is inhibited by p53 and further inhibits FABP4 and AdEV protein secretion from adipocytes. A: Differentiated 3T3-L1 adipocytes were treated with DMSO or 10 μmol/L nutlin for 48 h. Western blot analyses were used to measure intracellular phospho-S6 (Ser 240/244), phospho-S6 (Ser 235/236), S6, and actin levels. B: Differentiated 3T3-L1 adipocytes were treated with DMSO or 10 μmol/L DOXO for 24 h. Western blot analyses were used to measure intracellular Ser 240/244, Ser 235/236, S6, and actin levels. C: Differentiated 3T3-L1 adipocytes were treated with torin and rapamycin for 24 h. Western blot analyses were used to measure intracellular protein Ser 240/244, Ser 235/236, S6, NAMPT, FABP4, and actin levels, as well as secreted protein levels (NAMPT, FABP4, CD63, and TSG101) in the cell culture medium. For the intracellular lysate, whole-cell lysates were harvested for each condition and equal amounts of intracellular protein were loaded to Western blots. For the secretion analyses, equal volumes of cell culture medium were loaded onto Western blots. For the AdEV markers CD63 and TSG101, AdEVs were pelleted from equal volumes of cell culture medium by using Total AdEV isolation reagent. The pellets were lysed and resuspended in equal volumes of RIPA buffer before loading onto Western blots. D: Differentiated 3T3-L1 adipocytes were treated with torin and rapamycin for 24 h. AdEVs were pelleted, purified from an equal volume of cell culture medium from different treatments, and resuspended in 1 mL PBS for future analyses. AdEV size and amounts were measured by Nanosight analyses. All results represent the mean ± SEM. *P < 0.05, **P < 0.01. EV, extracellular vesicle.