Figure 2

Ability of CRKL to regulate cell proliferation in the MKN74 gastric cancer cell line. (A) siRNA knockdown for CRKL in MKN74 cells with CRKL amplification. Cells were reverse-transfected with the siRNA oligonucleotides (20 nM) using HiPerFect Transfection Reagent, and 8 × 10⁵ cells were seeded in 60 mm-dishes with 4 ml media. The expression of CRKL was examined 4 days after the reverse transfection of CRKL siRNA or negative control siRNA using a western blot analysis with anti-CRKL monoclonal antibody (Y244; 1:500 dilution). The level of CRKL protein expression was decreased in CRKL siRNA-transfected cells, compared with mock-transfected cells. The expression of β-tubulin protein was analyzed as an internal control. (B) Decrease in the proliferation of MKN74 cells transfected with siRNA for CRKL. Cells were reverse-transfected with the siRNA oligonucleotides (20 nM), and 1.5 × 10⁴ cells were seeded in 96-well microplates containing 100 μL of media. After the reverse transfection of CRKL siRNA or negative control siRNA, the number of viable cells was counted by measuring the reduction in the tetrazolium monosodium salt WST-8. The cell number of CRKL siRNA-transfected cells relative to that of mock-transfected cells is shown. Values are the mean ± standard deviation of three independent experiments. P-values were calculated using the t-test, and * indicates a statistical significance.