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. 2021 Mar 30;124(12):1959–1969. doi: 10.1038/s41416-021-01354-5

Fig. 2. 5-ASA suppresses β-catenin/TCF transcriptional activity.

Fig. 2

a, b TOPflash reporter assay at 24 h after 20 and 40 mM 5-ASA. a PC/AA/C1 adenoma and b LS174T CRC-derived cells. Mean ± SEM; n = 3; *p < 0.05; **p < 0.01 c (i) Western blots of PC/AA/C1 and LS174T at 24, 48 and 72 h after treatment with 20 and 40 mM 5-ASA showing expression of active dephosphorylated and total β-catenin protein; α-tubulin was used as the loading control. (ii) Densitometry graphs show the fold change of active dephosphorylated β-catenin protein as a ratio of total β-catenin expression over the 72-h period. 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. d Western blot of LEF-1 expression in PC/AA/C1 and LS174T cells to determine the specificity of the LEF-1 antibody. The expression level of LEF-1 was measured by western blotting 72 h after transfection with a LEF-1 SMARTpool siRNA or negative control. The results are representative of three independent experiments. β-Actin was used as the loading control. e (i) Western blot showing PC/AA/C1 and LS174T cells after 24, 48 and 72 h after treatment with 20 and 40 mM 5-ASA. Wnt/β-catenin target proteins AXIN-2, c-MYC and LEF-1 are all downregulated with 5-ASA, with the most marked phenotype observed at 72 h. β-Actin was used as the loading control. (ii) Densitometry graphs show the expression change of AXIN-2, c-MYC and LEF-1 as a fold of the loading control at the 72-h 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; ***p < 0.001.