Fig. 3.

Overexpression of mCherry-tagged seipin greatly reduces TAG synthesis and formation of LDs. Cells were transfected with empty or seipin-expressing pmCherry-N1 plasmids for 48 h before assays. A: Transfected HeLa cells were treated with 200 μM oleate for another 16 h and cellular TAG amount was analyzed subsequently. Right panel in A, relative cellular TAG content was normalized to 75% transfection rate [Normalized relative TAG content = (Relative TAG content-25%)/75%]. * P < 0.01, compared with empty vector control. B: Transfected HeLa and NIH3T3 cells were treated with 200 μM oleate for another 16 h. Cells were stained with BODIPY 493/503 and observed for LDs. Bar, 10 μm. C: Seipin overexpression did not affect fatty acid uptake. Transfected HeLa cells were incubated in the presence of 2 µg/ml BODIPY FL C₁₆. At 1 h or 16 h, cells were fixed with 4% formaldehyde and observed for the distribution of BODIPY fluorescence. Bar, 10 μm. D: Seipin overexpression did not enhance TAG lipolysis. Transfected HeLa cells were further treated with 200 μM oleate for another 16 h. Free glycerol released into culture media was analyzed as described in Materials and methods. E: Seipin overexpression reduced oleate incorporation into TAG. Transfected HeLa cells were pulsed with [¹⁴C]oleic acid for 2 h. Incorporation of [¹⁴C]oleic acid into TAG was normalized to protein concentrations and 75% transfection rate. *, P < 0.01. F: Seipin overexpression does not affect TAG synthesis in vitro. Microsomes were prepared from transfected HeLa cells and incubated in the presence of 0.4 mM dioleoylglycerol and 2.5 nmol [¹⁴C]oleoyl-CoA (120,000 dpm/nmol) for 10 min. Lipids were separated via TLC and [¹⁴C]TAG formed was quantified by scintillation counting. G: Seipin overexpression does not significantly change the expression level of key lipogenic enzymes. Fold changes of the mRNA levels of genes involved in TAG synthesis (seipinS-mCherry/vector).