Extended Data Figure 4: Mechanically induced phospho-Tyr654-β-catenin by tumour growth pressure do not co-localize with E-cadherin but with villin.
From: Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure
a, Top, Ki67 expression under UML plus magnet conditions show increased number of positive cells per crypt, in contrast to controls (without UML, or without magnet with UML, measured in n = 2 mice, 33 crypts, for each condition. Blue is DAPI. Bottom, P values are of P < 0.001 between any individual conditions. Statistical test is the non-parametric exact Mann–Whitney test for all preceding quantitative experiments. b, No co-localization of pTyr654-β-catenin with sub-apical junctional E-cadherin in response to tumour growth pressure and co-localization with villin, in Apc+/1638N mice with magnetic pressure (measured in n = 2 mice by condition). c, No co-localization of pTyr654-β-catenin with sub-apical junctional E-cadherin in response to tumour growth pressure and co-localization with villin, in wild-type mice with magnetic pressure (measured in n = 2 mice by condition). d, Immunofluorescence analysis of pY1062-Ret kinase in response to ex vivo mechanical strain. The phosphorylation of Ret Y1062 is observed in ex vivo Apc+/1638N colon upon mechanical compression (Methods) (54.26 ± 16% positive crypts out of 236, measured in n = 7 mice), initiating at 1 min compression and compared to control (0.6 ± 1.3% of positive crypts out of 99, measured in n = 5 mice). Mechanical activation of pY1062-Ret is impaired by wide range PP1 and PP2 Src-family kinase inhibitors (1.55 ± 0.25% positive crypts out of 142 and 6.03 ± 3.5% positive crypts out of 192, respectively, measured in n = 2 mice for each condition). Sunitinib, a Ret inhibitor (9.85 ± 3.8% positive crypts out of 143, measured in n = 4 mice), blocks mechanical activation of pY1062-Ret and SU6656, a Src-specific inhibitor (39.5 ± 8.5% of positive crypts out of 95, measured in n = 3 mice), does not block mechanical activation of pY1062-Ret. e, Ex vivo mechanical induction of pY654-β-catenin after 20 min (measured in n = 2 mice) (upper panel), enrichment of cytoplasmic and nuclear β-catenin after 20 min (measured in n = 3 mice) (middle panel), and Myc expression after 4 h (measured in n = 6 mice) (lower panel), impaired by Sunitinib inhibitor treatment (measured in n = 2 mice by condition) in Apc+/1638N mice. β-catenin control: 13.97 ± 2.7% positive crypts out of 121, measured in n = 2 mice; compressed: 69.82 ± 8.7% positive crypts out of 119, measured in n = 2 mice; compressed + sunitinib: 24.13 ± 1.2% positive crypts out of 119, measured in n = 2 mice. Myc control: 10.96 ± 0.21% positive crypts out of 118, measured in n = 2 mice; compressed: 75.08 ± 2.8% positive crypts out of 148, measured in n = 6 mice; compressed + sunitinib: 23.36 ± 1.1% positive crypts out of 60, measured in n = 2 mice. Ex vivo mechanical compression of Apc+/1638N mice tissues caused an increase of β-catenin-positive nuclei (14.35 ± 5 a.u. (arbitrary units), measured in n = 2 mice) by a factor of 3.6 compared to the control (3.97 ± 1.9 u.a, measured in n = 2 mice), impaired in the presence of Sunitinib (4.9 ± 1.9 a.u., measured in n = 2 mice). Merged images of DAPI and β-catenin were obtained with ImageJ co-localization analysis on ten images analysed with 4–6 crypts per image. White spots represent a positive co-localization. f, No co-localization of pTyr654-β-catenin with sub-apical junctional E-cadherin and co-localization with villin in Notch/Apc mice (measured in n = 2 mice). Here, the original colour for E-cadherin and villin was blue to avoid Notch–GFP green fluorescence, and was changed to green for the colour coherence of the complete figure. g, No co-localization of pTyr654-β-catenin with sub-apical junctional E-cadherin and co-localization with villin, in Apc+/1638N ex vivo compressed mice. Some apical E-cadherin is observed (measured in n = 2 mice).
