Figure 2.

Survivin is expressed upon insulin exposure through the PI3K/mTOR pathway in mature adipocytes. During the differentiation process of 3T3-L1 cells and C3H10T1/2 cells, Survivin mRNA and protein levels were determined by qRT-PCR (a and c) and western blot (b and d). Survivin expression was detected by qRT-PCR and western blot in 3T3-L1 adipocytes, which were treated with 100 nM insulin for the indicated time (e) and insulin at the indicated concentrations for 24 h (f). Primary adipocytes, isolated and cultured from the SVF of subcutaneous fat, were induced to differentiate into mature adipocytes. Then Survivin content was determined by western blot after treating with 100 nM insulin for the indicated time (g) and insulin at the indicated concentrations for 24 h (h). Survivin content was immunobloted after rapamycin (10 nM) and LY294002 (50 μM) for one hour prior to insulin exposure (100 nM) both in 3T3-L1 adipocytes and primary adipocytes (i). (j) 3T3-L1 adipocytes were incubated with insulin (100 nM) for 24 h, then incubated with or without HBSS for the indicated time. Immunoblots determined the levels of Survivin and GAPDH. (k) Protein levels of Survivin were detected by western blot in 3T3-L1 adipocyte overexpressing Survivin after incubation with HBSS for the indicated time. (l) The stability of ectopic expression of Survivin was evaluated in a cycloheximide (CHX, 100 μg/ml) chasing experiment. Cells were harvested 0, 15, 30 or 60 min after the addition of CHX. The ectopic expression of Survivin protein level was evaluated using western blot. *P<0.05, **P<0.01, ***P<0.001