Figure 1. MLN4924 attenuates CRL4^DCAF1 activity and reduces the proliferation of NF2-mutant schwannoma cells.

(A) 293T cells transfected with Flag-HA-tagged DCAF1 (FH-DCAF1) were treated with the indicated concentrations of MLN4924 for 4 hours and then lysed in RIPA buffer. Input and FLAG immunoprecipitates were immunoblotted as indicated. (B) COS-7 cells were transfected with FH-DCAF1 and Myc-Ubiquitin as indicated and then treated with MLN4924 at the indicated concentrations for 4 hours. FLAG immunoprecipitates and input were immunoblotted as indicated. Red asterisk indicates putative auto-ubiquitylated DCAF1. Red arrowhead indicates putative CRL4^DCAF1 substrates. (C) In vivo Lats1 ubiquitylation assay. 293T cells were transfected as indicated. 24 hours following transfection, cells were treated with MLN4924 (8 hours as indicated) and the proteasome inhibitor MG132 (4 hours, all samples) prior to lysis in a denaturing buffer. Input and Ni-NTA precipitates were immunoblotted as indicated. (D) FC-1801 cells were treated with the indicated concentrations of MLN4924 for 8 hours, lysed, and immunoblotted as indicated. (E) The Nf2-null mouse schwannoma line FC-1801 and the Nf2-wild-type immortalized adult Schwann cell line FH-912 were treated with increasing doses of MLN4924 and subjected to MTT proliferation assay after 72 hours of treatment. Proliferation of MLN4924-treated cells was normalized to respective DMSO-treated controls. The concentrations of MLN4924 required to inhibit cell proliferation by 50% (GI₅₀) are indicated.