Fig 2: Effect of siRNA-mediated knockdown of genes co-amplified with MET or ERBB2 on cell apoptosis or cell cycle progression(A) Cell cycle analysis using flow cytometry and (B) western blot analysis of apoptosis markers in SNU620 cells treated with CAPZA2 or MET siRNA. (C) Cell cycle analysis using flow cytometry and (D) western blot analysis of apoptosis markers in NCI-N87 cells treated with siRNAs targeting ERBB2 or genes co-amplified with ERBB2 (GRB7, MIEN1, PGAP3, or STARD3). For cell cycle analysis, the percentage of cells at each phase is shown as the mean ± SD of at least three experiments. Mann-Whitney test, *P < 0.05, **P < 0.01.

Fig 3: Validation of CNAs and gene expression of genes co-amplified with MET or ERBB2 in TCGA data(A) OncoPrint of CNAs in CAPZA2 and genes co-amplified with ERBB2 in 295 GC samples based on TCGA data [16]. (B, C) Box plot showing the association between their mRNA levels and gene amplification. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and the whiskers indicate the highest and lowest values of the results. OncoPrint was generated using the cBioPortal (http://www.cbioportal.org/). Statistical differences between the two groups were assessed using the Mann-Whitney test. The number of patients for each group (in parentheses) is provided.

Fig 4: Effect of siRNA-mediated knockdown of genes co-amplified with MET or ERBB2 on proliferation of GC cells(A,B) Effect of MET or CAPZA2 knockdownon proliferation of diffuse-type GC cell lines SNU620 and SNU5. (C,D) Effect of knocking down ERBB2, GRB7, MIEN1, PGAP3, or STARD3 on proliferation of intestinal-type NCI-N87 and SNU216 cells. The protein products of target genes before (ctrl) and after siRNA treatment were measured by western blotting in a time-dependent manner (48, 72, and 96 h). Depletion or marked reductions in target protein levels in siRNA-treated cells compared with before siRNA treatment were confirmed (the exception was STARD3). Significant knockdown of STARD3 at the mRNA level was confirmed by qRT-PCR (data not shown). The number of viable control siRNA (siCTRL)- or target gene siRNA (siCAPZA2, siMET, siERBB2, siGRB7, siMIEN1, siPGAP3, siSTARD3)-transfected cells was measured at the indicated times after siRNA treatment. Data are presented as the mean ± standard deviation (SD) of three or four experiments. Mann-Whitney test, *P < 0.05.

Fig 5: Association between expression of genes co-amplified with MET or ERBB2 and overall survival in patients with GCKaplan-Meier plot of overall survival (OS) for GC patients stratified by gene expression of (A) CAPZA2 and MET, and (B) ERBB2 and genes co-amplified with ERBB2 (GRB7, MIEN1, PGAP3, and STARD3). Patients were divided into low expression and high expression groups according to cut-off value, and statistical differences in survival between the two groups were tested using the log-rank test. The number of patients in each group and the number of events (death) are provided. HR: hazard ratio.