Fig. 6: CREPT assists β-catenin retaining in the crypt nuclei.

a–d CREPT deletion impaired the SuperTop luciferase activity stimulated by Wnt1 (a), APC depletion (b), β-catenin (c), and CHIR99021 (d). CREPT was deleted by a CRISPR-Cas9 system in HEK293T cells. SuperTop-luciferase reporter and pRL-TK plasmids transfected WT and CREPT deleted HEK293T cells were co-transfected with Wnt1 (a), shAPC (b), and β-catenin (c) plasmids, or treated with 5 μM CHIR99021 for 4 h (d). The activity was expressed as fold-changes, normalized by an internal control (Renilla). p < 0.0001 (a). p = 0.0168 (b). p = 0.0009 (c). p = 0.0002 (d). n = 3 independent experiments for each treatment. e Endogenous CREPT interacts with β-catenin. Cell lysis of intestinal crypts was incubated with control IgG or anti-CREPT antibodies. The immunoprecipitants were analyzed by Western Blotting using anti-β-catenin or anti-CREPT antibodies. f Exogenous CREPT interacted with β-catenin. Myc-tagged CREPT (Myc-CREPT) and FLAG-tagged β-catenin (Flag-β-catenin) were co-expressed in HEK293T cells. g CREPT deletion reduced the nuclear protein level of β-catenin. CREPT was deleted by the CRISPR-Cas9 system in DLD1 colorectal tumor cells. β-catenin proteins were analyzed by Western blotting in cytoplasmic and nuclear fractions of WT and CREPT KO DLD1 cells. h–k Representative images of β-catenin IHC staining in crypts at 0, 1, 3, and 5 dpi after X-ray irradiation, respectively. Obvious nuclear β-catenin appeared at 3 dpi of WT mice, but not Vil-CREPT^KO mice. l Quantification of nuclear β-catenin positive crypts per 1 mm intestine at 3 dpi. p < 0.0001. n = 3 mice per genotype. Images are representative of at least three independent experiments (n = 3 mice per genotype). Statistics data represent mean ± SEM. All p values were generated by 2-tailed Student’s t-test. Scale bars: 20 μm (h–k).