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

Figure 5.

Figure 5

Glypicans mediate the differential effects of NOTUM activity in Apc mutant vs Apc/Trp53 mutant mouse tumouroids. (A) Brightfield image of Apc mutant mouse tumouroids (A) and Apc/Trp53 double mutant tumouroids (AP) infected with control sgRNA (sgCtl), Gpc1 sgRNA (sgGpc1), Gpc4 sgRNA (sgGpc4), Gpc6 sgRNA (sgGpc6) (n=3 technical replicates). Scale bar: 100 µm. (B) EdU assays quantifying fraction of cells in S-phase from cultures shown in (A) (n=3 technical replicates). (C) Western blotting in Apc mutants infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. Note multiple β-ACTIN blots, one for each of three identical replicate blots. (D) Western blotting in Apc/Trp53 mutant tumouroids infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. (E) Western blotting for GPC1 (left) and GPC4 (right) using N-terminal antibodies recognising the extracellular domain of indicated glypicans in the media supernatant (Med) or cell lysate (Lys) of Apc mutant or Apc/Trp53 double mutant tumouroids infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. (F) Western blotting in Apc mutant tumouroids infected with control sgRNA (sgCtl) or Gpc1 sgRNA (sgGpc1). β-ACTIN was used as a loading control. (G) Western blotting in Apc/Trp53 mutant tumouroids infected with control sgRNA (sgCtl) or Gpc4 sgRNA (sgGpc4). β-ACTIN was used as a loading control. (H) Coimmunoprecipitation of IGF1Rb with anti-GPC1 antibody in Apc mutant tumouroids in the presence or absence of the small molecule NOTUM inhibitor ABC99. (I) Coimmunoprecipitation of TGFβR1 with anti-GPC4 antibody in Apc/Trp53 double mutant tumouroids in the presence or absence of the small molecule NOTUM inhibitor ABC99. (J) Western blotting assessing non-canonical TGFβ-TAK1-p38a pathway activity in APKS tumouroids in response to NOTUM inhibition. β-ACTIN was used as a loading control. (K) AP and APKS tumouroids treated with the p38 inhibitor SB202190, with EdU-based quantification of S-phase (n=3 technical replicates). Scale bar: 100 µm. For all panels: **p<0.01, ***p<0.001, Student’s t-test. See online supplemental figures 8,9 for validation of driver mutations, shRNA/sgRNA knockdown and overexpression.