Figure 5: Peristalsis drives Wnt-independent β-catenin activation in KRAS mutant cells.

(A) Heat map of Wnt ligand gene expression. Gene expression changes in peristalsis for HCT116 KRAS^MUT, RKO KRAS^WT and RKO KRAS^MUT cells are demonstrated as fold-change values compared to static controls (indicated by 1.0). Statistical differences between each cell type exposed to peristalsis and static controls individually can be found in Supplementary material (Supp. Fig. 6). (B) Representative flow cytometry analysis graphs and box and whisker plot of flow cytometry LGR5 expression (%) following 24 hour exposure to peristalsis or static conditions with Wnt inhibition (WNTi). HCT116 KRAS^MUT and RKO KRAS^MUT resulted in sustained increases in LGR5 expression in peristalsis compared to static controls (**p<0.01, *p<0.05, two-way ANOVA). (C) Representative micrographs of cells maintained as static controls or exposed to peristalsis with and without Wnt inhibition (WNTi) stained with β-catenin (magenta) and counterstained with DAPI (blue). Scale bar 10 μm. Bar graphs quantifying nuclear β-catenin localization relative to their respective static controls for all tested conditions. Peristalsis increased nuclear localization of β-catenin compared to static controls both with and without Wnt inhibition in HCT116 KRAS^MUT and RKO KRAS^MUT (****p<0.0001, t-test). RKO KRAS^WT cells exposed to peristalsis with and without Wnt inhibition did not observe increases in β-catenin nuclear localization. Detailed quantification methods and channel separated images are found in supplementary information (Supp. Fig. 2 and 4).