Figure 4

Responses of the MKN74 gastric cancer cell line with CRKL amplification to treatment with BMS354825 (a dual Src/BCR-ABL kinase inhibitor) and CRKL-targeting peptide. (A) Viability of MKN74 cells treated with BMS354825 but not those treated with AMN107 (a highly selective BCR-ABL kinase inhibitor) is decreased. The cells were seeded in 96-well microplates at a density of 1 × 10⁴ per well; after 24 h, the drug (0.01–1.0 μM) or 0.1% DMSO solution was added. Viability was examined in the MKN74 cells after 72 h of treatment at the indicated concentration using WST-8 reagent. The number of viable cells after treatment with each inhibitor was normalized to the number of viable cells without treatment, and the relative viability is shown in the graph. Values are the mean ± standard error. P values were calculated using the Dunnett’s multiple comparison test, and * indicates a statistically significant decrease. (B) Effective inhibition of CRKL phosphorylation in MKN74 cells treated with BMS354825. Cells were treated with each inhibitor (DMSO only or 0.01 μM of drug) for 90 min, and the expression of CRKL protein was examined using a western blot analysis with anti-phospho CRKL polyclonal antibody (Y207; 1:1,000 dilution) or anti-CRKL monoclonal antibody (Y244; 1:500 dilution). The expression of β-tubulin protein was analyzed as an internal control. (C) Comparison of CRKL mRNA transcripts between AGS and MKN74 cells using real-time QRT-PCR analysis. The amounts of CRKL transcripts normalized to the amount of GAPDH transcripts are shown in the graph. (D) Comparison of expression of CRKL protein between AGS and MKN74 cells using a western blot analysis. The expression of CRKL was examined using the primary antibodies shown in (B). The expression of β-tubulin protein was analyzed as an internal control. (E) Detection of CRKL gene copy number in AGS cells using a FISH analysis. The CRKL signal is red, and the control signal for chromosome 22 is green. Nuclei are stained with DAPI. (F) Viability of AGS cells decreased after BMS354825 treatment. Viability was examined as described in (A). Values are the mean ± standard error. P values were calculated using a t-test, and * indicates a statistically significant decrease. (G) MKN74 cells with CRKL amplification and AGS cells without CRKL amplification were seeded in 96-well microplates at a density of 1 × 10⁴ per well. 24 h after seeding, cells were treated with CRKL-targeting peptide (0.0925–25 μM) or 0.2% DMSO solution at the indicated concentration. The sequence of the CRKL-targeting peptide that was used is shown above the graph. After 72 h of incubation, viability was determined using an MTT assay. The results are presented as the mean ± standard deviation of three independent experiments. P values were calculated using a t-test, and * indicates a statistically significant difference between the cells treated with CRKL-targeting peptide and those treated with DMSO. (H) Cell proliferation of MKN74 and AGS cells treated with CRKL-targeting peptide (6.25 μM) or DMSO as measured by counting cells using a hemocytometer. Cells (1 × 10⁴) were seeded in 24-well plates and treated with CRKL-targeting peptide or DMSO. The cell counting was performed every 24 h for 3 days. Data are shown as the mean ± standard deviation of three independent experiments. P values were calculated using a t-test, and * indicates a statistically significant difference between the cells treated with CRKL-targeting peptide and those treated with DMSO.