Figure 5

Suppression of downstream signaling and cell growth in KRAS -amplified gastric cancer cells by siRNA-mediated knock-down of KRAS. Cells were transfected with siRNA targeting KRAS, LRMP, CASC1 or LYRM5, or a non-targeting siRNA as a negative control. (a) KRAS knock-down suppressed the phosphorylation of p44/42 in KRAS-amplified cells. Protein levels of KRAS and activated p44/42 48 h post-transfection were determined by immunoblot using anti-KRAS and anti-phospho-p44/42 antibodies. The status of gene amplification and mutation (codon 12) of KRAS in each cell line is indicated. +, presence; -, absence. (b) KRAS knock-down suppressed the phosphorylation of p44/42 MAP kinase and AKT in MKN1 cells. Twenty-four h after siRNA transfection, MKN1 cells were cultured for an additional 24 h in regular medium (Nontreated), serum-starved for 24 h (Starved) or serum-starved then stimulated with 10% FCS for 1 h (+Serum). The activation of p44/42 and AKT was determined by immunoblot using phospho-specific antibodies. (c) Suppression of cell growth in KRAS-amplified cells by KRAS knock-down. Cells were transfected with siRNA, and cell number at the indicated time points after transfection was determined indirectly by WST-8 colorimetric assay. Data is presented as percent decrease in cell number as compared to cells transfected with control siRNA at each time point, and represents the means and SD for triplicate cultures. Statistical analysis was performed using the unpaired t-test. *, P < 0.005 relative to the siRNA control. Data is representative of two independent assays. (d) KRAS knock-down decreased the fraction of HSC45 and AGS cells in S-phase. Cells were analyzed by flow cytometry 48 h post-transfection. Data is representative of two independent assays.