Figure 5.

Inhibition and over-activation of β-catenin transcriptional activity alters pressure response

(A) Max z projections and orthogonal view of pre-confluent, confluent, and folded (left to right) fixed MDCK monolayers immunostained with anti-YAP-A568.

(B) Normalized YAP intensity ratio (nuclear YAP intensity normalized with mean nuclear YAP intensity in non-folded regions) of non-folded and early and late folded capsules (see schematic). See also Figure S4.

(C) Total cell lysates from flat versus MDCK cells in 2.5% alginate capsules were analyzed by western blotting. Tubulin was used as loading control.

(D–H) Representative confocal midplane image of FUCCI cells in 2.5% alginate capsules treated with 25 μM iCRT3 (D) and quantification of mean cell density (cells/um²) over time (E), mean pressure (kPa) over time (F), mean FDCs over time (G), and mean FDCs over mean pressure (kPa) (H) for control (circle, blue) and iCRT3-treated (triangle, purple) FUCCI cells within 2.5% capsules.

(I–K) Representative confocal equatorial plane images of FUCCI cells in 2.5% capsules from −10 to 20 h from 100 nM BIO treatment (I) with quantification of mean cell number (J) and mean relative FDCs (K) for control (circle, blue) and BIO-treated (triangle, green) FUCCI cells in 2.5% capsules. Inset in (J) shows mean cell density (cells/um²) over time. Relative FDCs refers to FDCs normalized to FDCs at 12.5 h prior to drug treatment. Differences between means in (K) are ns with a p value of 0.40. For (B), N = 3 capsules and n < 120 cells per condition, error bars are SEM. For (E)–(H), control n = 20 and iCRT3-treated n = 12; statistical significance measured using t test with Welch’s correction p < 0.05. For (J), control N = 8 capsules, BIO-treated capsules N = 4; statistical significance was measured using one-way ANOVA with Dunnet’s test, at p < 0.05, samples were assumed to be different. Scale bars, 100 μm.