Fig. 6: FIM-04-806 induces ANLN degradation by inhibiting USP10 deubiquitinase activity.
From: Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma

A Effect of F806 on ANLN expression was evaluated in xenografts (KYSE510 cells) from tumor-bearing mice. After F806 (4 mg/kg) treatment for 21 days, the level of ANLN protein in xenografts was detected by immunohistochemistry. B Cells were treated with F806 (10 μM) for 15 h and then co-treated with MG132 (10 μM) for 6 h, ANLN levels were detected by western blotting. C Cells were co-treated with cycloheximide (CHX) (10 μg/ml) and F806 (10 μM), and ANLN levels were examined by western blotting. D KYSE150 cells transfected with HA-Ub were treated with F806 (10 μM) for different times and then treated with MG132 (20 μM) for 8 h before harvest. Ubiquitination assays were performed to examine the ubiquitination level of ANLN. E KYSE150 cells transfected with Flag-vector or Flag-USP10 were treated with F806 (10 μM), and ANLN levels were detected by western blotting. F KYSE150 cells transfected with HA-USP10 or HA-USP10-CA were treated with F806 (10 μM), and ANLN levels were detected by western blotting. G KYSE150 cells were transfected with USP10 or control siRNA for 24 h and then treated with F806 (10 μM) for 16 h, and ANLN levels were detected by western blotting. H HEK293T cells were transfected with the indicated plasmids for 24 h, then treated with MG132 (10 μM) with or without F806 (10 μM) for 6 h. Subsequently, cells were harvested for ubiquitination assay. I KYSE150 cells were treated with F806 (10 μM) for 24 h. Cell lysates were subsequently labeled with HA-Ub-VS for 30 min. The indicated antibodies were used for the western blotting. J Purified USP10 protein was incubated with F806 at 25 °C for 2 h in vitro, and then labeled with HA-Ub-VS for 30 min. The indicated antibodies were used for the western blotting. K, L USP10 purified from prokaryotic cells and different concentrations of F806 were mixed at 25 °C for 2 h. Ub-AMC was then added to each well and further incubated at 37 °C for 1 h. Ub-AMC hydrolysis was measured in real time (K), and the biochemical IC50 of F806 was measured using GraphPad Prism 7 (L). All data are representative of at least three independent experiments and the results were statistically analyzed using a t-test.