Fig. 6
Nur77 is the target of DdBIC. Melanoma A375 cells were treated with DdBIC (20 μM) for 0.5 h to display Nur77 mitochondrial localization and its interaction with other proteins, for 4 h to measure the levels of heme and mito-ROS, for 8 h to assess pyroptotic features, unless otherwise specified. a Nur77 was knocked down to detect pyroptosis. b DdBIC bound to a pocket formed by helices 5, 8, and 10 of the LBD. The LBD is shown as a light blue cartoon, amino acids near DdBIC are shown as sticks, and hydrogen bonds are shown as yellow dashed lines. c Measuring the binding affinity of DdBIC with either the LBD or LBD4mt via ITC. The titration data were analyzed via the program MicroCal PEAQ-ITC Analysis Software and fitted with a one-site binding model. The levels of LBD or LBD4mt were indicated (right). d Cells were treated with DdBIC for the indicated times, and the expression levels of Nur77 were detected. e Cells were treated with DdBIC to observe Nur77 localization. Nur77 and mitochondria were indicated with their corresponding antibodies, and nuclei were shown by DAPI staining. f Cells were transfected with HK2 and Nur77, and their interaction was determined (top). Nur77 was transfected into HK2-knockdown cells, and the mitochondrial localization of Nur77 was determined (bottom). g HK2 was knocked down to detect pyroptosis. h Binding surface of the LBD for HK2. The binding of DdBIC to the LBD induces conformational changes in residues Q528, R563 and E445 (white sticks) from those in the apo structure (purple sticks). i Nur77 and SDHA were overexpressed, and the mitochondrial fraction was prepared to determine the interaction between Nur77 and SDHA. j The levels of mitochondrial heme and mito-ROS were determined in Nur77-knockdown cells. k Nur77 and HK2 were separately knocked down in cells, and OMA1 activity, OPA1 cleavage, phosphorylation of PERK and eIF2α, and GZMB expression levels were analyzed. Statistics: two-way ANOVA with Tukey’s test to a, g and j. P values are shown
