Figure 7.

Indisulam induces ZEB1 degradation through the ubiquitin proteasome system.A, proteasome inhibitor MG132 abolishes the indisulam-induced ZEB1 degradation. AGS and MGC803 cells were pretreated with DMSO or indisulam (10 μM) for 48 h and then treated with DMSO or MG132 (10 μM) for 16 h in the presence of DMSO or indisulam. The resulting cell lysates were subjected to immunoblotting analysis. Mean ± SD (n = 3), Student’s t test, ∗∗p < 0.01, ns: not significant. B, DCAF15 knockdown eliminates the indisulam-induced degradation of ZEB1. AGS cells stably expressing shNC and shDCAF15 were treated with DMSO or indisulam (10 μM) and cycloheximide (200 μg/ml) for the indicated time. Mean ± SD (n = 3), two-way ANOVA, ∗∗p < 0.01, ns: not significant. C, indisulam promotes ZEB1 ubiquitination. HEK293T cells were transfected with the indicated plasmids for 48 h, pretreated with MG132 (10 μM) for 2 h, and treated again with DMSO or indisulam (10 μM) for 12 h in the presence of MG132. ZEB1 was immunoprecipitated with anti-HA magnetic beads. The cell lysates and immunoprecipitates were subjected to immunoblotting analysis. The relative intensity of ubiquitinated ZEB1 was shown below the image. The experiments were performed twice and similar results were obtained. DMSO, dimethyl sulfoxide; HA, hemagglutinin.