Fig. 1
Identification of DKC1125, which suppresses KITENIN gain of function in vitro, by screening a small-molecule library and detection of KSRP, which binds specifically to DKC1125. a Structure of DKC1125 and its inhibitory effect on cell invasiveness. After suspending 7 × 104 empty vector-transfected Caco2 CRC cells (Caco2/EV) or KITENIN-transfected cells (Caco2/KITENIN-V5) in medium containing 0.2% BSA, a Transwell invasion assay was performed. Cells were exposed to the compound for 48 h at the indicated concentrations (up to 10 μM). Images represent three independent experiments. The histogram represents invading cells, which were counted in five chosen areas, calculated as rate relative to vehicle-treated control, and represented as bar graphs (mean ± SEM, n = 3). The asterisk indicates a significant difference between Caco2/KITENIN-V5 cells (vehicle-treated vs DKC1125-treated; *P < 0.05; **P < 0.01) and Caco2/EV cells (vehicle-treated vs DKC1125-treated; #P < 0.05). b Structure of chemical probe containing DKC1125. The chemical probe was constructed by covalently attaching Affigel beads to the DKC1125 linker. Cell invasion assay was performed in HCT116 CRC cells, comparing DKC1125-TN-linker-acetate and DKC1125 itself. DKC1125-TN-linker-acetate (DKC1125-Linker-Ac) was constructed by attaching an acetate group to the linker instead of Affigel at the same position as DKC1125, and was used as a positive control for Affigel-bound DKC1125. c Detection of KSRP protein through analysis of pull-down proteins with the chemical probe. The chemical probe, prepared as described above, was mixed with the HCT116 cell lysate for pulldown of the chemical-binding proteins, which were verified by immunoblot analysis using antibody against KSRP and KITENIN (upper panel). Immunoprecipitation (IP) was performed with KSRP-specific antibody, and elevated KITENIN binding was observed in the presence of DKC1125 (0.5 μM) (lower panel). d Assessment of the downstream effectors of the KITENIN axis after treatment of Caco2 cells with DKC1125 (0.5 μM). e Effects of modulation of KSRP on the invasiveness of Caco2 and HCT116 cells, and influence of DKC1125 treatment. Forty-eight hours after transfection of each construct, the increase in invasiveness due to KSRP expression was examined by Transwell assay in si-scrambled (si-NC) or si-KITENIN-expressing HCT116 cells (left panel) or elevated invasiveness by KITENIN expression in si-scr or si-KSRP-expressing Caco2 cells (right panel) with or without DKC1125 (0.5 μM). Data are expressed as in Fig. 1a. *p < 0.05, compared with the control group