Fig. 4: Cisplatin binds to C622 in GR that mediates nuclear translocation and transcriptional activation.

a Interaction between GR and cisplatin was determined by surface plasmon resonance (SPR) analysis and shown as a dissociation constant value. GR protein was enriched from 239T cells carrying pLHCX-flag-GR. b Differential scanning fluorimetry of GR incubated with increasing concentrations of cisplatin. c Cellular thermal shift assay using KB-3-1 cells treated with vehicle, cisplatin, or dexamethasone. d Coomassie-stained SDS-PAGE gel of purified recombinant GR ligand-binding domain variants, GR-LBDm WT, C622A, C643A, and C736A. e Interaction between GR-LBDm variants and cisplatin is determined by SPR analyses. f, g Effect of cisplatin and dexamethasone on GR nuclear translocation in KB-3-1 expressing GR WT or C622A mutant. KB-3-1 cells were treated with 1 μg/ml cisplatin or 500 nM dexamethasone for 24 h followed by immunofluorescence assay (f) or nuclear/cytosol fractionation (g). β-actin and PARP were used as cytoplasmic and nuclear markers, respectively. c: cytosol, n: nucleus. Scale bars represent 10 μm. h, i Effect of cisplatin and dexamethasone on MAST1 promoter activity in KB-3-1 expressing GR WT or CA mutants (h) or in KB-3-1 and A2780 cells expressing GR WT or C622A mutant (i). Compounds were administered as described in f. Data are from one biological experiment for a, and mean ± SD from three independent biological experiments for the others. Statistical analyses were performed by one-way ANOVA for g–i. Source data are provided as a Source Data file.