Additional file 6: of The oncogene KRAS promotes cancer cell dissemination by stabilizing spheroid formation via the MEK pathway

Figure S3. Assessment of apoptosis in ID8 and ID8-KRAS cells in vitro and in vivo. a ID8 and ID8-KRAS cells (1 × 106) were incubated for 48 hours in 2D or 3D culture. Floating and attached cells were collected, washed with PBS, and subjected to PI/Annexin-V staining. Annexin V-FITC (5 μl) and propidium iodide (5 μl, 50 μg/ml) were added to the cell suspension. The stained cells were analyzed and the percentage of PI-negative/Annexin-V-positive apoptotic cells was measured by flow cytometry. Experiments were repeated at least three times. The values shown represent the mean ± SEM (*p < 0.05). b,c Mice were i.p. injected with ID8-GFP or ID8-KRAS-GFP cells (1 × 106). Peritoneal washes were collected 24 hours later. ID8-GFP and ID8-KRAS-GFP cells were collected by centrifugation, washed with PBS, and subjected to Annexin-V staining. The stained cells were analyzed by flow cytometry. A quantitative analysis of the percentage of the GFP-positive cancer cells in total cells obtained from peritoneal washes (b) and the percentage of apoptotic cells in GFP-positive cancer cells (c). The values shown represent the mean ± SEM (*p < 0.05, n = 6 mice per group). (PDF 29 kb)