Fig. 4: 5-ASA reduces the ability of PC/AA/C1 adenoma and LS174T carcinoma cells to form spheroids.

a–e PC/AA/C1 human adenoma-derived cells. a Mean number of spheroids in each well 7 days after culture. n = 3 ± SEM; *p < 0.05. b Mean log spheroid area over 21 days in culture. c Log spheroid area of spheroids at day 21, demonstrating distribution of spheroid size. n = 1 ± SD representative of n = 3. d Mean log spheroid area of spheroids at day 1. n = 3 ± SEM; *p < 0.05; **p < 0.01. e Representative images of PC/AA/C1-derived spheroids at day 21. Images acquired using Leica DM16000 microscope, ×5 lens with the Leica LAS-X software. Images were processed using MatLab software. f–i LS174T human carcinoma-derived cells. f Mean number of spheroids in each well 7 days after culture. n = 3 ± SEM; ***p < 0.001. g Mean log spheroid area over 21 days in culture. h Log spheroid area of spheroids at day 21, demonstrating distribution of spheroid size. n = 1 ± SD representative of n = 3. i Mean log spheroid area of spheroids at day 21. n = 3 ± SEM; **p < 0.01; ***p < 0.001. j–k Quantitative PCR (QPCR) mRNA analysis of LGR5 and CD133 gene expression. j PC/AA/C1- and k LS174T-derived spheroids after 21 days of treatment with 5-ASA. All mRNA values are normalised to the housekeeping genes TBP or HPRT. Data show relative mRNA quantity of LGR5 and CD133 presented as a fold change of the control, which itself was normalised to one. Data are presented as the mean of three independent experiments ± SEM; n = 3. One sample t test was used to determine statistical significance, *p < 0.05; **p < 0.01. dRn baseline-corrected normalised fluorescence.