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. 2010 Jan 20;285(13):9604–9615. doi: 10.1074/jbc.M109.043786

FIGURE 3.

FIGURE 3.

Fsp3K-A enhances endogenous Fsp27 stability and promotes lipid storage in adipocytes. A, the expression of Fsp3K-A in differentiated 3T3-L1 adipocytes enhances the stability of endogenous Fsp27. Differentiated 3T3-L1 cells (day 6 after induction) were infected with an adenovirus encoding HA-tagged Fsp3K-A or GFP as a negative control. Two days after infection, cells were harvested 0, 15, 30, or 60 min after the addition of CHX. B, the quantitative analysis of the level of endogenous Fsp27 from A. Experiments were repeated three times. **, significant difference from control curve using a two-way ANOVA, p < 0.01. C, knockdown of Fsp27 in 3T3-L1 adipocytes using lentivirus-delivered siRNA against Fsp27 and overexpression of HA-Fsp27, HA-Fsp3K-A, or GFP in Fsp27-deficient 3T3-L1 adipocytes mediated by adenovirus infection. A lentivirus generated from the empty vector, which encoding only GFP, was used as a negative control. D, increased Fsp3K-A stability in Fsp27-deficient 3T3-L1 adipocytes. Differentiated Fsp27-deficient 3T3-L1 adipocytes were infected with an adenovirus encoding HA-tagged wild-type Fsp27 or Fsp3K-A. Two days after infection, cells were harvested at the indicated time points after the addition of CHX for stability analysis. E, Fsp3K-A promotes lipid storage in adipocytes. Fsp27-deficient 3T3-L1 adipocytes were infected with an adenovirus encoding HA-Fsp27, HA-Fsp3K-A, or GFP for 2 days, and the total cellular lipids were extracted. The cellular TAG content was evaluated using TLC. A Student's two-tailed t test (unpaired) was used for statistical analysis (*, p < 0.05; ***, p < 0.001, n = 3).