NR-V04 induces a ternary complex formation and mediates NR4A1 degradation through the ubiquitin-proteasome system. (A) PLA showing ternary complex formation induced by NR-V04. CHL-1 (left panels) and A375 (right panels) cells were pretreated with 0.5 μM MG132 for 10 min and then treated with DMSO, 500 nM celastrol, or 500 nM NR-V04 for 16 h. Representative images were shown for PLA assay (20× magnification); two experiments. Bar represents 50 μm for all images. (B) Co-IP experiment showing complex formation between NR4A1 and VHL by NR-V04 treatment. Co-IP was performed in NR4A1-Flag overexpressed HEK293T cells that were pretreated with 0.5 μM MG132 for 10 min, followed by 16-h treatment with DMSO or 500 nM NR-V04. NR4A1 was pulled down using an anti-Flag antibody conjugated to magnetic beads; two experiments. (C) NR-V04 induces NR4A1 degradation via VHL E3 ligase- and proteasome-dependent manner. CHL-1 cells were pretreated with 0.5 μM MG132 or 10 µM VHL 032 for 10 min, followed by 16-h treatment with DMSO or 500 nM NR-V04, three experiments. Source data are available for this figure: SourceData F5.