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. Author manuscript; available in PMC: 2020 Mar 10.
Published in final edited form as: Gastroenterology. 2019 Nov 16;158(4):1000–1015. doi: 10.1053/j.gastro.2019.11.019

Figure 4. p-AKT is reduced in TTC7A-deficiency.

Figure 4.

(A) Histopathology analysis of p-AKT in human colon tissue. Co-staining of cytokeratin 20 (CK20), a marker for the intestinal epithelium, and p-AKT is present in the normal and IBD control, while p-AKT is diminished in TTC7A-deficiency patients. Patient 1 is the colonoid donor and Patient 2 is unpublished with confirmed biallelic mutations. RedDot 2 nuclear counterstain (blue). Sections were magnified at 20× objective. (B) Histopathology analysis of pan-AKT (total AKT) in human colon tissue. Biopsies are the same as described in Figure 5A. Pan-AKT is present in all samples albeit with reduced intensity in the TTC7A-deficiency patient samples. Sections were magnified at 20× objective. (C) Immunoblot for p-AKT, XIAP, and cleaved Caspase 3 in WT and TTC7A-KO cells. After 3 h leflunomide treatment in TTC7A-KO cells, p-AKT and XIAP protein levels are detectable, while cleaved Caspase 3 is diminished. DMSO (*) (n=3). (D) Densitometric analysis of p-AKT, XIAP, and cleaved Caspase 3 from WT and TTC7A-KO cells. One-way ANOVA with post hoc test (Tukey), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, (n=3). (E) ttc7a+/− and ttc7a−/− whole mount zebrafish staining with p-AKT (red), Synaptic vesicle protein 2 (SV2) (green), and RedDot 2 nuclear counterstain (blue). SV2 staining, indicating neuromuscular junctions, is absent from the intestinal epithelial monolayer and aids in differentiating epithelial cells from other nearby cell types. In the DMSO treated panel, p-AKT staining (Ser473) in ttc7a+/− fish is evident along the gastrointestinal tract, while absent in ttc7a−/− fish. Leflunomide treatment (3–7 dpf) restores p-AKT staining in the intestinal epithelium of ttc7a−/− fish. Fish were magnified at 5× objective, (n=4).