Fig. 6

DMOCPTL regulated ubiquitination by directly binding to GPX4. a Docking of DMOCPTL into the active site of GPX4 (PDB code: 5H5S). The protein structure is shown as a cartoon diagram with selected residues labeled (left). Surface representation of the protein residues (Middle). 2D representation of binding interaction of DMOCPTL and GPX4 (right). b The IC50 values of DMOCPTL probes in MDA-MB-231 cells after the treatment for 72 h by MTT assay. c The effect of DMOCPTL and probe 8a on the viability of MDA-MB-231 cells after treatment for 72 h. d The scheme for synthesis of probes 8a–8e. e The scheme for synthesis of probe 10. f The workflow of probe 10 for identifying DMOCPTL-interacting proteins by in vitro pull-down assay in MDA-MB-231 and SUM159 cells. g MDA-MB-231 and SUM159 cell lysates were incubated with probe 10 for 2 h at room temperature. After incubated with streptavidin conjunct agarose A + G beads, the bead-bound proteins were collected and detected by western blot experiments. h The workflow of probe 8a for identifying DMOCPTL-interacting proteins by in situ pull-down assay in MDA-MB-231 and SUM159 cells. i MDA-MB-231 and SUM159 cells after the treatment of probe 8a for 6 h were collected and performed click reaction to conjugate biotin with probe 8a. After incubated with streptavidin conjunct agarose A + G beads, the bead-bound proteins were collected and detected by western blot experiments. j The scheme for synthesis of probe 10a. k The co-localization of GPX4 (green), Ubiquitin (purple), probe 10a (red); the nucleus DAPI (blue) in MDA-MB-231 cells by immunofluorescence assay. l MDA-MB-231 cells after the treatment of probe 8a for 6 h were collected and performed click reaction to conjugate biotin with probe 8a. After incubated with streptavidin conjunct agarose A + G beads, the bead-bound proteins were collected and analyzed by sliver staining