Figure 4.

Indisulam enhances the interaction between ZEB1 and DCAF15.A, DCAF15-interacting proteins were obtained from BioGRID (https://thebiogrid.org/). The cutoff threshold for the modified CompPASS score (https://bioplex.hms.harvard.edu/comppass/) is >0.75. The score for ZEB1 is 1.00. B, ZEB1 interacts with DCAF15. HEK293T cells were transfected with FLAG-DCAF15 and/or hemagglutinin (HA)-ZEB1 plasmids and then split into two plates for 48 h. Cells were pretreated with MG132 (10 μM) for 2 h and lysed. ZEB1 was immunoprecipitated with anti-HA magnetic beads. Cell lysates and immunoprecipitates were subjected to immunoblotting analysis. C, indisulam enhances the DCAF15-ZEB1 interaction. FLAG-DCAF15 plasmid or control vector was transfected into HEK293T cells for 48 h. Cells were pretreated with MG132 (10 μM) for 2 h and then treated with dimethyl sulfoxide (DMSO) or indisulam (10 μM) for 14 h. Cells were lysed, and DCAF15 and its interacting proteins were immunoprecipitated with anti-FLAG affinity gel. Cell lysates and immunoprecipitates were subjected to immunoblotting analysis. The relative intensity of the immunoprecipitated ZEB1 was shown below the image. D, ZEB1 colocalizes with DCAF15 in the nucleus. HEK293 cells were transiently transfected with the control or indicated plasmids for 24 h, fixed, and incubated with the primary and fluorescent secondary antibodies. Immunofluorescence was detected under a confocal microscope. The scale bar represents 5 μm.