Extended Data Figure 8: Notch/Apc colon shows an activation of the Ret/β-catenin/Myc mechanotransductive signalling pathway one month after tumour growth initiation.
From: Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure
a, Top, Ret Y1062 phosphorylation (measured in n = 4 mice, 317 crypts), apical β-catenin Y654 phosphorylation (measured in n = 4 mice, 223 crypts), cytoplasmic and nuclear β-catenin enrichment (measured in n = 2 mice, 16 images analysed with 4–6 crypts per image), and Myc expression activation (at 2.5 months, measured in n = 2 mice, 194 crypts), in Notch/Apc tumorous tissue compared to non-tamoxifen-injected Notch/Apc mice controls (measured in n = 4 mice, 264 crypts; measured in n = 4 mice, 198 crypts; measured in n = 2 mice, 18 images analysed with 4–6 crypts per image; measured in n = 2 mice, 315 crypts, respectively). GFP fluorescence (green) reveals a nuclear overexpression of Notch in the tumorous crypts. Note that at 1 month after tamoxifen injection, GFP expression is often diffuse and found in the nuclei and cytoplasm. Immunofluorescence staining and ImageJ co-localization analysis revealed an enrichment of nuclear β-catenin (white and purple spots represent a positive co-localization between β-catenin (red) and DAPI (blue)) by a factor of 5.8 in Notch/Apc colon samples 1 month after tamoxifen injection (11.64 ± 2.5 a.u., measured in n = 2 mice), compared to the control (1.99 ± 0.8 a.u., measured in n = 2 mice). Bottom, phosphorylation of β-catenin Y654 in Notch-negative crypts in tamoxifen-injected crypts (measured in n = 4 mice, 68 crypts, yellow arrows) compared to non-tamoxifen-injected control conditions (measured in n = 4 mice, 198 crypts). Cytoplasmic enrichment and nuclear translocation of β-catenin in Notch-negative crypts (measured in n = 4 mice). Nuclear translocation of β-catenin is assessed by co-localization with DAPI (in white) in Notch-negative crypts of Notch/Apc tissues after 1 month of tamoxifen injection compared to non-tamoxifen-injected control conditions. White spots and purple represent a positive nuclear DAPI and β-catenin co-localization, with a preference for the peripheral privileged sites of transcriptionally active chromatin. Immunofluorescence staining and ImageJ co-localization analysis revealed an enrichment of nuclear β-catenin (white and purple spots represent a positive co-localization between β-catenin (red) and DAPI (blue)) by a factor of 15 in Notch-negative crypts in colon samples 1 month after tamoxifen injection (33.6 ± 8.7 a.u., measured in n = 2 mice), compared to the control (1.99 ± 0.8 a.u., measured in n = 2 mice). Ten images with 4–6 crypts per image were analysed in each condition. Scale bars are 10 μm. b, Quantification of a. P < 0.001 in all cases, Student’s t-test.
