Ckx41 32 inverted microscope

For the colony formation assay, cells were seeded in 6-well plates at a density of 500 cells/well and cultured in complete medium (Thermo Fisher Scientific, Inc.). Two weeks later, the cells were fixed and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 30 min at room temperature. The number of visible colonies (>50 cells/colony) was counted using the CKX41-32 inverted microscope (magnification, ×100; Olympus Corporation).

For the cell invasion analysis, a 24-well Transwell assay plate (Corning Life Sciences) was used. Matrigel (Sigma-Aldrich; Merck KGaA) was diluted with serum-free RPMI-1640 medium (1:3) and used to coat the upper surface of the Transwell chamber at room temperature for 1 h. RCC cells (2×10⁴ cells/well) exposed to different treatments were added into the upper chamber with 500 µl serum-free RPMI-1640. The lower chamber was filled with 500 µl complete medium with 20% FBS, and cells were cultured for 48 h at 37°C. Subsequently, the invasive cells were fixed with methanol and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) at room temperature for 1 h. Images of cell invasion were captured under the CKX41-32 inverted microscope (Olympus Corporation).

Caki-1 and 786-O cells were seeded at a density of 1×10⁶ cells/well in 6-well plates and cultured in medium with a low concentration of FBS (5%). FBS was used in order to reduce cell proliferation risk, cells were cultured in a medium (19 (link)). After culture for 24 h when cells reached ~90% confluency, they were scratched using a 200-µl pipette tip and carefully washed with sterile PBS twice to remove the debris. Then, cells were treated with various doses of CK (10, 20 or 40 µM) at 37°C. At different time points (0 and 24 h), the images were captured using the CKX41-32 inverted microscope (Olympus) and the results were analysed using ImageJ software (v1.46; National Institutes of Health).