Fig. 7: Antagonism of the Wnt pathway by UBE2T inhibitor M435-1279 in GC.

A The effect of M435-1279 on ubiquitination of RACK1. Lysates from 293T cells expressing the indicated plasmids after 48 h treated with M435-1279 (11.88 μM) were immunoprecipitated (IP) with an anti-Flag followed by immunoblotting against indicated antibodies. B The effect of M435-1279 on the interaction of UBE2T with RACK1. M435-1279 did not affect UBE2T-Rack1 interaction by immunoprecipitated assays. Lysates from 293T cells expressing the indicated plasmids after 48 h treated with M435-1279 (11.88 μM) were immunoprecipitated (IP) with an anti-Flag followed by immunoblotting with indicated antibodies. C The effect of M435-1279 on the total or nuclear β-catenin expression of HGC27 cells. Forty-eight hours after M435-1279 treatment, total cell lysates (bottom), cytoplasmic, or nuclear lysates (top) were analyzed by western blot with the indicated antibodies (Lamin B1 as a nuclear loading control, GAPDH as a cytoplasmic loading control). D–F Effect of M435-1279 treatment on tumor (D) proliferation and (E, F) migration in HGC27, AGS, and MKN45 cells. Proliferation and migration ability were detected by colony-formation assay and Transwell assay, respectively. Student’s t test was used to examine statistical significance (mean ± S.D., n = 3, **P < 0.01, *P < 0.05). G–K MKN45 cells were intratumor injected in nude mice. The compound was dosed by intratumor injection at a single dose of 5 mg/kg/day, and DMSO was used as a control group. Shown are (G) representative image, (H) tumor weights, (I) tumor volumes, and (J) body weights. K The representative immunohistochemical images of Ki-67, RACK1, and β-catenin in intratumor tumors of mouses. Scale bar, 40 μm. Student’s t test was used to examine statistical significance (mean ± S.D., n = 15, **P < 0.01, *P < 0.05).