Consequence of Menin Deficiency in Mouse Adipocytes Derived by In Vitro Differentiation

Figure 3

3T3-L1 cells with menin knockdown can also undergo in vitro differentiation into adipocytes and show increased adipocyte cell size. (a) In vitro adipogenesis. Control shRNA plasmid (C-) or Men1 shRNA plasmid (M-) transfected 3T3-L1 cells were plated on day 1 in 3T3-L1 cell propagation medium overnight. The medium was replaced with differentiation medium for 2 days, followed by maintenance medium for 4 days to produce adipocytes, with a medium change (maintenance medium) every two days. (b) Menin knockdown. Whole cell extracts prepared from 3T3-L1 cells differentiated as shown in (a) were analyzed for the level of menin knockdown by western blot (day 8 adipocytes). Antitubulin was used as the loading control. Percent knockdown of menin was calculated after normalization to tubulin. Data are shown for 3 independent experiments. (c) Expression of adipocyte marker genes. RNA isolated from the 3 independent experiments shown in (b) was analyzed for the indicated adipocyte marker genes (Adiponectin, Pgc1α, and Pparγ) and an internal control gene (Gapdh) by conventional RT-PCR followed by agarose gel electrophoresis. Lanes marked “undiff” are 3T3-L1 cells before in vitro differentiation. (d) Oil Red O staining. Bright-field microscopy images of C- and M- differentiated 3T3-L1 cells stained for lipids/triglycerides after adipogenesis by Oil Red O staining show round adipocyte cells with lipid droplets (orange colored spots). Images are shown for experiment 2. Magnification = 400x. (e) Relative cell size. Cell diameter was measured as an index of cell size from 3 to 5 microscopy fields using the cell outline formed by the circle of orange lipid droplets accumulated inside the round adipocytes from the 3 independent 3T3-L1 differentiation experiments performed in (b) (an example marked with a black circle and arrow is shown in (d)). Diameter mean and SD are shown (). Note: the units on the -axis do not reflect the actual diameter of the cells but they reflect the measurement of the cell diameter from images captured at 400x magnification. (f) Relative lipid content. After adipogenesis, Oil Red O staining for lipids/triglycerides was performed, dye was extracted, and the OD was measured at 520 nm. Relative lipid level is shown for 2 independent experiments performed in (b) (OD at 520 nm). Error bar = SD. . (g) Relative expression of differentially expressed genes identified by microarray analysis of mESC-derived adipocytes. Genes more than 5-fold downregulated () or upregulated () in mESC-derived menin-null adipocytes were analyzed by QPCR using RNA isolated from adipocytes derived by in vitro differentiation of 3T3-L1 cells shown in (b) (experiment 2). QPCR relative fold change (normalized to Gapdh) is shown for 4 genes. The other 16 genes did not yield a PCR product in either cell type (C- or M-). (>2-fold change in M- versus C-).

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