Fig. 4

Treatment with DKC1125 results in autophagic degradation of Dvl2 and KITENIN through increased binding to RACK1. a RACK1 directly binds to DKC1125. Proteins pulled down by chemical probe using HCT116 lysates were verified by immunoblot analysis using antibodies against RACK1 and Dvl2. b Interactions of RACK1 with KSRP or Dvl2 within the functional KITENIN complex following DKC1125. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5 (KIT-V5), and treated with vehicle (V) or DKC1125 (DKC) (0.5 μM). The cell lysates were immunoprecipitated with anti-RACK1 antibody and immunoblotted with anti-KSRP or anti-Dvl2 antibody. c Levels of Dvl2 and KITENIN are reduced more after DKC1125 treatment under RACK1 expression. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5, or co-transfected with KITENIN-V5 and RACK1-GFP, and then treated with vehicle or DKC1125 (0.5 μM). The protein levels of Dvl2 and KITENIN were checked after treatment with cycloheximide at the indicated times. d RACK1 affects the activation of canonical WNT signaling by Dvl2. 293 T cells were transfected with the TOP-flash reporter gene and Dvl2, RACK1, or si-RACK1, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Differences in transcriptional activity of TCF/LEF by Dvl2, alone or in combination with RACK1 overexpression or knockdown, were compared between the presence or absence of DKC1125. e Autophagic degradation of Dvl2 and KITENIN by DKC1125. Caco2 cells were initially pretreated with vehicle, the proteasome inhibitor MG132 (MG, 10 μM), the lysosomal degradation inhibitors bafilomycin A1 (A1, 100 nM) and chloroquine (CQ, 10 μM), or the autophagosome blocker type III phosphatidylinositol 3-kinase inhibitor (3-MA, 1 mM), and later treated with a high concentration of DKC1125 (5 μM). Levels of Dvl2 and KITENIN were examined by immunoblot analyses. f Staining of autophagosomes in DKC1125-treated cells. Autophagosomes were stained in stably KITENIN-expressing Caco2 cells after 24 h treatment with DKC1125 using CYTO-ID autophagy detection dye. Rapamycin was included as a positive control for autophagic induction