Fig. 3. 5-ASA suppresses the expression of the stem-marker LGR5.

a Endogenous levels of LGR5 expression in a panel of colorectal adenoma- and carcinoma-derived cell lines. PC/AA/C1, S/AN/C1, S/RG/C2 colorectal cells, PC/AA/C1/SB10 transformed adenocarcinoma cells, HT29, HCA7, HCT116, HCT15, SW480, SW620, LOVO, LS174T colorectal adenocarcinoma cells and SW837 and SW1463 rectal adenocarcinoma cells were grown to ~70% confluence before collection of total protein for western blot analysis. α-Tubulin used as the loading control. b (i) Western blot analysis demonstrating downregulation of LGR5 in three adenomas (PC/AA/C1, S/AN/C1 and S/RG/C2), left, transformed adenocarcinoma cells (PC/AA/C1/SB10) and two CRC-derived cell lines (LS174T and SW620), right, 24, 48 and 72 h after treatment with 20 and 40 mM 5-ASA. β-Actin was used as a loading control. LGR5 is highly glycosylated⁵⁹, leading to the different banding patterns seen in the different cell lines. (ii) Densitometry graphs show the expression change of LGR5 as a fold of the loading control at the 72h timepoint. Expression is normalised to the respective control. Data are presented as the mean ± SEM of three independent experiments. n = 3. One sample t test was used to determine statistical significance. *p < 0.05; **p < 0.01. c (i) Western blots of LGR5 expression in PC/AA/C1 adenoma cells demonstrating downregulation of LGR5 after commencing treatment with 20 and 40 mM 5-ASA, but a subsequent reversal of this regulation once 5-ASA was withdrawn. β-Actin was used as a loading control. (ii) Western blot analysis of LGR5 expression in the 12 h after stopping 5-ASA treatment. β-Actin was used as a loading control.