Figure 4.

3D spheroid images at different concentration and time points A), and five‐point dose–response curves B) increasing concentration from 30 to 100 µM (5‐(3′,4′‐dihydroxyphenyl)‐γ‐valerolactone) on HCT116 EGFP spheroids in aggregation for 48, 120, 168, and 216 h. Two‐way ANOVA followed by Tukey multiple comparison test revealed a time‐dependent efficacy: 30 µM significant differences between 48 h time point versus 120, 168, and 216 h time points (p < 0,0001), 50 µM significant differences between 48 h time point versus 120 h (p < 0.0001), 168 h (p < 0.01), and 216 h (p < 0.0001) time points, and 70 µM significant differences between 48 h time point versus 120 h (p < 0.05), 168 h (p < 0.001), and 216 h (p < 0.0001) time points, and between 120 h time point versus 216 h time point (p < 0.05). The time‐dependent efficacy was also calculated using 5‐FU (10 µM) and DMSO at 0.05% C). In this case, the two‐way ANOVA followed by Tukey's multiple comparison tests revealed that 5‐FU significantly affected SI between 48 h time point versus 168 and 216 h time points (p < 0.05). SI was quantified by High Content Imaging System Operetta (Perkin Elmer) and expressed as a percentage of SI of vehicle‐treated controls at 48 h (DMSO 0.05%). IC₅₀ was calculated applying dose–response‐inhibition nonlinear regression analysis. Data are expressed as mean ± SEM. All experiments were performed from at least four independent experiments. Spheroids were imaged on High Content Imaging System Operetta (Perkin Elmer) with 10XLWD objective in brightfield and green fluorescent channels (merged images) at different concentrations. Scale bars, 200 µm.