Figure 1.

Menin-null (Men1-KO) mouse embryonic stem cells (mESCs) can undergo in vitro differentiation into adipocytes and show increased adipocyte cell size. (a) In vitro adipogenesis. Feeder-free mESCs (WT or Men1-KO) were allowed to form embryoid bodies (EBs) in medium without LIF for 2 days, followed by retinoic acid treatment for 3 days. On day 5, retinoic acid induced EBs were cultured in gelatin-coated plates with adipocyte differentiation medium for 21 days to obtain adipocytes, and the medium was replaced every 2 days. (b) Expression of adipocyte marker genes. Conventional RT-PCR followed by agarose gel electrophoresis of the adipocyte marker genes (Adiponectin, Pgc1α, and Pparγ), Men1, and an internal control gene (Gapdh) using RNA samples before and after in vitro differentiation of WT or Men1-KO (KO) mESCs into adipocytes. (c) Oil Red O staining. Bright-field microscopy images of WT and Men1-KO mESCs stained for lipids/triglycerides after adipogenesis by Oil Red O staining show round adipocyte cells with lipid droplets (orange colored spots). Magnification = 400x. (d) Relative cell size. After adipogenesis, Oil Red O staining for lipids/triglycerides was performed, and images were captured by bright-field microscopy shown in (c). Cell diameter was measured as an index of cell size from 3 to 5 microscopy fields using the outline formed by the circle of orange lipid droplets accumulated inside the round adipocytes (an example marked with a black circle and arrow is shown in (c)). Diameter mean and SD are shown (^∗ p < 0.05). Note: the units on the y-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. (e) 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 (OD at 520 nm) is shown for adipocytes. Error bar = SD. ^∗ p < 0.05.