Seipin-A212P inhibits adipogenesis at early stages of adipocyte differentiation.

<p>(A) A schematic diagram of the constructs used in generating the Tet-on inducible cell lines. Both mouse Seipin wild type (Seipin-WT) and A212P Seipin mutant (Seipin-A212P) were tagged with Myc at the N-terminus, and co-expressed with mCherry. Expression of Seipin and mCherry was under the control of the TRE promoter, which was activated by the Tet-on protein in the presence of doxycycline (Dox). 3T3-L1 cells were first infected with virus co-expressing the Tet-on protein and EGFP to create Tet-on stable cells. These cells were then infected with virus containing TRE-WT or TRE-A212P to generate the 3T3-TRE-WT and 3T3-TRE-A212P inducible cell lines, respectively. (B) Tight regulation of Seipin expression by Dox treatment in 3T3-TRE-WT and 3T3-TRE-A212P cells at pre-adipocyte and mature adipocyte stages. Higher Seipin expression was achieved with increased amount of Dox treatment. Seipin was detected by Myc antibody. β-actin was used as a loading control. (C) 3T3-TRE-WT and 3T3-TRE-A212P cells were treated with 100 ng/ml of Dox at the indicated time points during adipocyte differentiation before they were stained with Oil Red O and photographed under a microscope at day 10. Scale bars = 200, 50, 200 and 50 µm, respectively, and apply to each row of images. (D) Lipid contents of 3T3-TRE-WT and 3T3-TRE-A212P cells were quantified by using Oil Red O extraction and normalized to the same type of cells without Dox treatment. Data are presented as mean ± SD from three independent experiments. ***p<0.001 vs. TRE-WT cells that were treated with Dox on the same day. (E-F) Expression of various inflammatory response markers (E) and adipogenic genes (F) were measured by real-time qPCR in 3T3-TRE-WT and 3T3-TRE-A212P cells treated with or without 100 ng/ml Dox at minus day 2 and harvested at day 4 post differentiation. Data are presented as mean ± SD from three independent experiments. Comparison was made between the same type of cells with or without Dox treatment. *p<0.05.