2.4. Cell proliferation, colony formation, soft agar, and Transwell assays

Cell proliferation assays were performed using the Cell Counting Kit‐8 (CCK‐8, Dojindo Laboratories, Kumamoto, Japan) in accordance with the manufacturer’s instructions. The indicated cells were seeded into 96‐well plates at a density of 1 × 103 cells/well. Subsequently, CCK‐8 solution (10 μL of CCK‐8 reagent in 90 μL DMEM) was added to each well, and the plates were incubated at 37°C for 1‐3 h. The optical density was measured at a wavelength of 450 nm. Soft agar and colony formation assays were performed to examine the viability and tumorigenicity of bladder cancer cell lines with OTUB1 knockout or overexpression. Briefly, 4 × 102 cells were seeded into 6‐well plates. After 2 wk of incubation, colonies were stained with 0.025% crystal violet at room temperature for 15 min, and images were captured using a scanner. For soft agar assays, 2 ml of 0.7% agar was plated into each well of 6‐well plates and then 1 mL of cells (2 × 104 cells) was mixed with 1 mL of 0.7% agar to form the upper gel. Plates containing cancer cells were incubated at 37°C in a 5% CO2 in air incubator for 2‐3 wk, then the number of colonies was counted, and images were captured under a microscope. For cell migration assays, Transwell inserts (Corning) were placed into 24‐well plates. DMEM with 40% FBS was added to the lower chamber and then the indicated cells were seeded into the upper chamber in serum‐free medium at a density of 4 × 104 cells/well. The plates were incubated for 48 h. Cells that migrated into the lower chamber were fixed with 4% paraformaldehyde and stained with 0.025% crystal violet, and images were captured under a microscope.

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