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. 2023 Aug 17;72(12):2294–2306. doi: 10.1136/gutjnl-2022-329140

Figure 3.

Figure 3

Tumour suppressive and oncogenic functions of NOTUM require catalytic activity and occur via paracrine/juxtacrine signalling. (A) Brightfield images of Apc-mutant mouse tumouroids infected with empty vector (EV), vector expressing wild-type NOTUM (OE) or catalytically dead NOTUM with Ser239Ala mutation (S239A) (n=3 technical replicates). Scale bar: 100 µm. (B) Clonal seed efficiency from single cells in (A). (C) S-phase/EdU incorporation assays in tumouroids from (A). (D) Brightfield images of the mouse APKS subclone with hypomorphic NOTUM mutation from online supplemental figure 1N,O (APKSHypo ) infected with EV, vector expressing wild-type NOTUM (OE), or catalytically dead NOTUM with Ser239Ala mutation (S239A) (n=3 technical replicates). Scale bar: 100 µm. (E) Clonal seed efficiency from single cells in (D). (F) EdU incorporation assays in APKSHypo organoid from (D). (G) Images of APKSHypo mouse tumouroids, with tumouroids infected with an mCherry-expressing vector and cocultured with tumouroids infected with a vector expressing GFP only (EV) or expressing GFP and NOTUM (OE) (n=3 technical replicates). Scale bar: 100 µm. (H) EdU assays in the cultures in (G), quantifying the percentage of cells in S phase in the GFP+or mCherry+populations or the overall population. *p<0.05,**p<0.01, ***p<0.001, Student’s t-test. see online supplemental figures 8,9 for validation of driver mutations, sgRNA knockdown and overexpression.