Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

Figure 2

Overexpression of CRACM1 inhibits MSC differentiation to adipocytes. MSCs, M1-MSCs, and KOM1-MSCs were cultured in adipogenic differentiation medium for 7 days. Cells were then stained for mFABP (red) as a marker of adipocytes and counterstained with Hoechst® 33342 for nuclear staining (blue). (a) Typical images of MSCs, M1-MSCs, and KOM1-MSCs observed by fluorescence microscopy (×200; scale bar: 50 μm). (b) Typical imaging screening panel for quantification of mFABP4 expression. MSCs, M1-MSCs, and KOM1-MSCs were seeded on 8-well plates, and 32 fields were captured in each well using a high-throughput image quantitation system. One of 32 fields is shown. Well number and average intensity are indicated on the image. (c) FABP expression in MSCs, M1-MSCs, and KOM1-MSCs. The average fluorescent intensity was obtained from 256 images for each group (). Results are expressed as . (d) Typical images of lipid droplets analysis. Lipid droplets, which were stained using Oil Red O, present as bright refractive round structures. (×400; scale bar: 25 μm). (e) Relative positive area of lipid droplet analysis. The red-stained area was segmented from the background, and the relative positive area was quantified. More than four fields per section and an average of five sections from each sample were used for semiquantitative analysis (). Results are expressed as .

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