Digital microscopic imaging system

Karyotype analysis of ES cells was performed as described previously [28 ]. Briefly, after cultured in feeder-free conditions for 24–36 h, ES cells were exposed to 10 μg/mL colchicine (Sigma, Kanagawa, Japan) for 1–2 h, and trypsinized into a single-cell suspension followed by suspension for 15 min in 0.075 mM KCl solution. Cells were repeatedly fixed in methanol/acetic acid mixture, then spread over slides and stained with Giemsa (Sigma, Kanagawa, Japan) solution. Karyotypes were observed and imaged under a Digital Microscopic Imaging System (Leica, DMIRB, Wetzlar, Gemany).

Cell invasion assays were performed using Transwell chambers with 8‐μm pore filters (Corning, New York, NY, USA). SMMC‐7721 and Bel‐7404 cells were seeded in six‐well plates and transfected with siSUMO2 or NC and incubated for 36 h. The cells were then seeded at a density of 5 × 10⁴ cells per well in 200 μL of serum‐free DMEM in the upper chamber; the bottom chamber contained 600 μL of DMEM with 10% FBS as a chemoattractant. After a 24‐h incubation, cells were fixed with methanol and stained with 0.1% crystal violet. Five random fields were photographed at ×200 magnification using a digital microscopic imaging system (Leica, Bensheim, Germany), and the cells within these fields were counted.