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. 2016 Aug 5;7:12321. doi: 10.1038/ncomms12321

Figure 6. Growth arrest of established murine tumours upon stromal Hh activation.

Figure 6

(a) Protocol schematic. (b) Example of a 3D μUS reconstruction; tumour volume (arrow) is highlighted in red, other hypoechoic (black) structure is the urinary bladder (ub); lu: colonic lumen, filled with US gel; vaw: ventral abdominal wall. (c) Tomato+ cells in an AOM/DSS-induced tumour from a Col1a2CreER;R26-LSL-tdTomato mouse 3 days after Tam administration; β-catenin (green) marks tumour cells; nuclear stain: 4,6-diamidino-2-phenylindole (DAPI); scale bar, 100 μm. (d) Tumour size as measured by μUS after Tam instillation (week 0). A total of n=5 Ptch1ΔCol1 mice with n=11 tumours and n=7 controls (littermates lacking Cre recombinase, floxed Ptch1 alleles or both) with n=12 tumours are included; 1 to 5 tumours per mouse were analysed. The adjusted P-values (fdr) from t-tests are indicated: P=0.406 (week 0), P=0.022 (week 1), P=0.029 (week 2), P=0.026 (week 3), P=0.026 (week 4) and P=0.078 (week 5). Box-and-whisker plots: whiskers represent minima and maxima. In the control group, mice had to be killed because of bleeding per anus, leaving 2 tumours for US analysis in week 5 and none in week 6. (e) Quantification of cleaved caspase-3 positivity per high-power field (original magnification × 400): controls (n=9 tumours), Ptch1ΔCol1mice (n=5 tumours), P=0.0034 (t-test). Middle and right panels show representative IHC images for similar-sized tumours 3 weeks after Tam treatment for the indicated genotypes. Scale bars, 20 μm. (f) Proliferative index as a percentage of Ki67+ nuclei for n=7 controls and n=4 tumours from Ptch1ΔCol1 mice, P=0.0467 (t-test) and representative images (middle and right panels). Scale bars, 50/20 μm. (g) ISH semi-quantification of Grem1 in control mice (n=5 tumours) and Ptch1ΔCol1mice (n=7 tumours). Stacked bars represent the frequency of intensity grading; P=0.0076 (Mann–Whitney test), middle and right panels show representative images for the two groups; scale bars, 200/100 μm. (h) Quantification of p-Smad1/5 IHC in control mice (n=8 tumours) and Ptch1ΔCol1 mice (n=9 tumours); P=0.0028 (t-test), middle and right panels show representative images for the two groups; scale bars, 50 μm; (e–h) boxed areas show magnified regions. *P<0.05, **P<0.01 and ***P<0.001.