Figure 1

Identification of highly amplified chromosome regions containing the CRKL gene and the detection of CRKL overexpression in gastric cancer. (A) Genome-wide detection of copy number alterations using a high-density SNP microarray in the MKN74 gastric cancer cell line. The copy number status for the whole genome of MKN74 is shown. DNA (250 ng) was analyzed using an Affymetrix GeneChip 250 K NspI array, and the total copy numbers were determined by analyzing the microarray data using the CNAG program. The chromosome number is shown above the panel. Chromosome 22 is highlighted in red. (B) The copy number status of chromosome 22 of the MKN74 cells is shown. A highly amplified region of chromosome 22 is enlarged, and the genes located in this region are indicated. The CRKL gene is highlighted in red. (C) Detection of CRKL amplification in MKN74 cells using a FISH analysis. The left panel shows the CRKL signal (red) in MKN74 cells, while the right panel shows the CRKL (red) in non-cancerous gastric tissue cells. An extreme increase in the CRKL copy number was observed in the MKN74 cells, while a normal copy number (2) was seen in non-cancerous cells. Nuclei are stained with DAPI. (D) Detection of the increased expression of CRKL mRNA transcript in 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. The average expression level of eight normal gastric mucosa samples was measured as a control. (E) Detection of the increased expression of CRKL protein in MKN74 cells using a western blot analysis. The expression of CRKL was examined using anti-CRKL monoclonal antibody (Y244; 1:500 dilution), horseradish peroxidase-coupled secondary antibody (1:5,000 dilution), and enhanced chemiluminescence detection reagents. The expression of β-tubulin protein was analyzed as an internal control.