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Fig. 3 | Molecular Cancer

Fig. 3

From: A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex

Fig. 3

DKC1125 inhibits the function of Dvl by interfering with the interaction of KSRP with Dvl2. a Association of KSRP with Dvl2 was inhibited after treatment of DKC1125. Co-IP experiments were performed in Caco2 cells stably expressing EV or KITENIN-V5 using cytoplasmic–nuclear fractionation after treatment with vehicle (V) or DKC1125 treatment (0.5 μM). Precipitates were analyzed by immunoblotting to detect KSRP-Dvl binding. Whole-cell lysate (WCL) of the same pool of cells was co-analyzed as a control. b The amount of Dvl2 protein adhering to KSRP was markedly reduced by treatment with DKC1125. Dvl2-ΔDIX-GFP transfected HCT116 cells were collected and lysed, and the supernatants were mixed with purified KSRP-His by Ni-NTA after in vitro bacterial expression and subjected to GFP-Trap. GFP-Trap, consisting of an anti-GFP Nanobody/VHH coupled to agarose beads, was used for effective pulldown of GFP-fusion proteins. Interaction of ΔDIX-DVL2-GFP with bacterially expressed KSRP-His was verified by immunoblotting with anti-His antibody. c Effect of DKC1125 on Dvl binding of WT-KSRP or ΔKH34-KSRP. Co-IP analysis was performed using cytoplasmic–nuclear fractions obtained from WT-KSRP or ΔKH34-KSRP transfected Caco2 cells stably expressing KITENIN-V5 or EV. Binding was verified by immunoblotting. d Effect of DKC1125 on activation of WNT/β-catenin by expression of KITENIN and/or KSRP. 293 T cells were transfected with the TOP-flash reporter gene and KITENIN-V5, WT-KSRP-myc, or ΔKH34-KSRP-myc, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Luciferase activity was measured at 48 h after transfection and normalized against the activity of TK-Renilla. Data are shown relative to the corresponding TOP-flash value in control cells. Differences in transcriptional activity of TCF/LEF by expression of KITENIN, WT-KSRP, and ΔKH34-KSRP were compared between the presence and absence of DKC1125. Error bars indicate SEM. The asterisk indicates a significant difference between groups (NS, not significant; **P < 0.01)

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