Fig. 4

Arginine supplementation rescues the cytostatic effect of F-IEX and L. lactis Lc4 CFS. (a) Confluence changes of HT-29 (top) and HCT116 (bottom) cells over time (0-72 h) after F-IEX (5% v/v) and vehicle (0.35 M NaCl + 10mM HEPES buffer – 5% v/v) treatment, with and without the addition of 5 mM L-arginine (ARG). Cell proliferation was monitored with the IncuCyte S3 live-cell analysis system. Scans were performed every 2 h. Each time point represents mean +/- SD, n = 3. (b) Growth rate constant of data presented in panel a. (c) Cell cycle analysis of HT-29 cells after 24 h of treatment with 5% (v/v) F-IEX and vehicle (same as in a) with the addition of 5mM L-arginine (ARG). Measured DNA content corresponds to the percentage of cells in each cell cycle phase (G0/1, S, G2/M). (d) Bar chart quantifying results from panel c. Data presented as mean +/- SD from n = 3 independent experiments. (e) 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% of F-IEX for 24 h, with and without the addition of 5mM L-arginine. 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. (f) The effect of 5 mM L-arginine addition on the cytostatic activity of Lc4 CFS evaluated on HT-29 cells with the MTS assay (*p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001 from one-sided t-test (b, d) or one-way ANOVA with Bonferroni correction (f)