Fig. 7: FIM-04-806 alters the balance of the USP10-ANLN-Cdh1 complex by targeting USP10 and inhibits the M/G1 transition of ESCC cells.

A The binding between F806 and USP10 was analyzed by SPR. His-USP10 protein was immobilized on an activated CM5 sensor chip, F806 solution was then flowed across the chip. B KYSE150 cells were transfected with Cdh1 or control siRNA for 24 h and then treated with F806 (10 μM) for 12 h, and ANLN level was detected by western blotting. C KYSE150 cells were treated with F806 (10 μM) for different times and MG132 (20 μM) for 8 h before harvest. The interaction between ANLN and Cdh1 or USP10 was detected by immunoprecipitation. D KYSE150 cells were synchronized in M phase and released for different time periods with DMSO or F806 (10 μM). The interaction between ANLN and USP10 or Cdh1 was detected by immunoprecipitation. E Cells were treated with different concentrations of F806 for 16 h. The indicated antibodies were used for the western blotting. F, G Experimental protocol for DTB. KYSE150 cells were synchronized by DTB and released for different time periods. F806 (10 μM) was added at 9 h after release. The samples were analyzed by flow cytometry (F) and immunofluorescence (G). H Cells 9 h after DTB release were treated with or without F806 (10 μM) for 3 h. The contractile ring localization of ANLN was also determined. Nine fields were counted for each group. I Cells after 9 h of release were treated with or without F806 (10 μM) for 6 h. The nuclear localization of ANLN was determined. Five fields were counted in each group. All data are representative of at least three independent experiments and the results were statistically analyzed using a t-test. J Model: USP10 and Cdh1 form a functional complex with ANLN in a non-competitive manner to regulate ANLN abundance. The steady state of ANLN levels promotes contractile ring assembly and cytokinesis. Moreover, F806 selectively targets USP10 to alter the ubiquitination-deubiquitination balance of ANLN. Reduction of ANLN leads to a slowdown in the turnover of contractile ring components and a delay in M phase.