Fig. 3

F-IEX fraction induces cell cycle arrest in the G0/1 phase, decreases c-Myc expression, and reduces phosphorylation of the p70 S6 kinase. (a) Confluence changes in HT-29 and HCT116 cells over time (0-72 h) after F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10 mM HEPES buffer – 5% v/v) or no treatment (control). Scans were performed every 2 h, each time point represents mean +/- SD (n = 3 independent experiments). (b) Growth rate constant of F-IEX-treated HT-29 and HCT116 cells. (c) Microscopic images from the IncuCyte S3 Live-Cell imaging system of HT-29 and HCT116 cells treated for 72 h with F-IEX (5% v/v) or vehicle (same as in a) ) as a control. (d) SYTOX Green-exclusion experiments. Green area-to-phase area ratio as quantification of changes in cells with disrupted membrane integrity during 72 h treatment with F > 50 kDa and GM17 > 50 kDa (5% v/v) and 5 µM of SYTOX Green (left graph) with corresponding confluence (right graph). Puromycin (0.5 µg/ml) was used as a cell death-inducing positive control. (e) Cell cycle analysis of HT-29 cells after 24 h treatment with 5% (v/v) F-IEX and vehicle (same as in a). (f) Bar-chart quantifying result from panel e. Data presented as mean +/- SD from n = 3 independent experiments. (g) Western blots showing c-Myc and phosphorylated p70-S6 kinase (P-p70-S6K) levels. HT-29 and HCT116 cells were treated with 5% vehicle (same as in a) or 5% (v/v) of F-IEX for 24 h. Total protein was isolated from the cells, resolved in SDS-PAGE gels, and analyzed using antibodies against indicated proteins. Actin was used as a loading control. n = 3 independent experiments. (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test)