Fig. 4

The conservative RNFL sequence in GSDME specifically interacts with the repeated WD40 domain of CDC20 A Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitation (IP) derived from 293T cells transfected with indicated constructs. Then cells were administered 10 µM MG132 for 12 h before harvesting. B Immunoblot analysis of whole cell lysates (Input) and IP derived from wild-type PC-3 cells. Then cells were administered 10 µM MG132 for 12 h before harvesting. C IB of WCL and His pull-down of PC-3 cells transfected with the indicated constructs. Then cells were treated with 10 µM MG132 for 12 h and lysates with denaturing buffer. D Schematics of indicated CDC20 constructs with or without the deletion of WD40 and Fizzy domains or N-term. E Molecular docking studies of GSDME full length against WD40 domain of CDC20. Protocol validation of molecular docking experiment using ZDock and PyMOL. Representative images of the predictive binding model were visualized. F IB of WCL and IP derived from 293T cells transfected with indicated constructs. G IB of WCL and His pull-down of PC-3 cells transfected with the indicated constructs. Then cells were treated with 10 µM MG132 for 12 h and lysates with denaturing buffer. H Sequence comparison of putative CDC20 binding motif in GSDME with known CDC20 substrates. CDC20 binding consensus sequences are shown in red. Schematics of indicated GSDME constructs with or without the RNFL consensus binding motif deletion. I The IB analysis of WCL derived from PC-3 cells transfected with the constructs of Flag-GSDME and HA-CDC20. J–K IB of WCL and IP derived from 293T cells transfected with indicated constructs. Then cells were treated with 10 µM MG132 for 12 h before harvesting. L PC-3 cells were transfected with the constructs as indicated. 30 h post-transfection, cells were treated with 100 µg/ml cycloheximide (CHX) and were harvested at the indicated time points