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Cells were plated, allowed to adhere overnight, and pretreated with tamoxifen or fulvestrant one hour prior to irradiation. Cells were harvested at indicated time points and lysed using RIPA buffer (ThermoFisher 89901) containing protease and phosphatase inhibitors (Sigma-Aldrich PHOSS-RO, CO-RO; Santa Cruz Biotechnology sc-3540, sc-24988A; Cayman Chemical 14333, 14405). Samples were separated on precast NuPAGE Bis-Tris protein gels (ThermoFisher), transferred to PDVF membranes (Millipore IPVH00010), and blocked with blotting grade blocker (BioRad 1706404, 5% milk). Antibodies used for protein detection include ERα (Cell Signaling 8644S; 1:1000), Rad51 (Millipore PC130; 1:500), β-actin (Cell Signaling 12262S; 1:50,000), total PARP1 (Abcam ab6079; 1:1000), cleaved PARP (Cell Signaling 5625S; 1:1000), cyclin E1 (Santa Cruz sc-247; 1:1000), cyclin B (Santa Cruz sc-245; 1:1000), and cyclin A (Santa Cruz sc-271682; 1:1000). Secondary antibodies used include anti-mouse (Cell Signaling 7076S; 1:10,000) and anti-rabbit (Cell Signaling 7074S; 1:10,000). All blots are derived from single experiments and are processed in parallel. Quantification of western blots was performed with ImageJ software.

Primary antibodies used: mouse anti-AIM-1 (1:100; 611082, BD Biosciences, Dublin, Ireland), rabbit anti-pSer7CENP-A (1:200; 04-792, Millipore, Cork, Ireland), anti-centromere (1:100; 15-234, Antibodies Incorporated, CA, USA), mouse anti-PLK1 (1:200 for IF; ab17057, Abcam, Cambridge, UK), mouse anti-PLK1 (1:500 for WB; sc-56948, Santa Cruz, Heidelberg, Germany), rabbit anti-Histone H3 pSer10 (1:50; 06-570, Millipore), rabbit anti-GAPDH (1:1000; 14C10 Cell Signaling, MA, USA), mouse anti-HEC1 (1:500 for IF, 1:1000 for WB; clone 9G2.23, Genetex, Eching, Germany), rabbit anti-ZWINT-1 (1:100 for IF, 1:1000 for WB; IHC-00095, Bethyl, TX, USA), mouse anti-pT210-PLK1 (1:300 for IF, 1:1000 for WB; 39068, Abcam), pT3H3 (1:400 for IF 1:1000 for WB; 9714S, Cell Signaling), pMPM2 (1:1000; 05-368, Millipore), anti-mouse Cyclin B (1:1000; SC-245, Santa Cruz). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse (1:300; A11001, Life Technologies, Dublin, Ireland), Alexa Fluor 546 goat anti-rabbit (1:300; A11010, Life Technologies), Alexa Fluor 647 goat anti-human (1:300; A21445, Life Technologies), goat anti-rabbit Alexa Fluor 488 (1:50 for FACS; A11008, Life Technologies), IRDye^® antibodies (1:10,000; LI-COR Biosciences, NE, USA).

For western blotting, whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.1% NP-40, 0.1% SDS) containing a protease inhibitor cocktail (BPI-9200, Tech & Innovation™, Bucheon, Korea) and a phosphatase inhibitor cocktail (45065; Santa Cruz, Santa Cruz, CA). Proteins were then separated on an 8% or 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes using an iBlotTM dry blotting system (Invitrogen). The transferred membranes were cut after the blocking steps to analyze with different antibodies. Immunoblotting analysis were performed with the following primary antibodies: pNEK9 (T210) (1:5000, ab63553; Abcam), NEK9 (1:5000, ab138488; Abcam), EG5 (1:1000, 4203; Cell signaling Technology), acetyl-α-tubulin (Lys40) (1:1000, T7451; Sigma-Aldrich), pAKT (S473) (1:1000, 9271; Cell signaling Technology), AKT (1:1000, 9272; Cell signaling Technology), cyclin B1 (1:1000, sc-245; Santa Cruz) and β-Actin (1:10,000, 5441; Sigma-Aldrich). Rabbit or mouse horseradish peroxidase (HRP)-labelled secondary antibodies (1:10,000; ENZO, Farmingdale, NY) were then used. The blots were visualized using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific-Pierce, Rockford, IL).

Cell lysis and immunoblotting were performed as described [55 (link)]. Proteins were detected using anti-ACC, anti-pSer⁷⁹-ACC, anti-AMPKα2, anti-pThr¹⁷²-AMPK, anti-FASN, anti-Cyclin B1 (sc-245, Santa Cruz, Biotechnology, CA, USA), anti-pHistoneH3 (#9701, Cell Signaling), anti-GFP (G6539, Sigma) and anti-α-tubulin. GAPDH and PCNA were detected with anti-GAPDH (GTX100118, GeneTex) and anti-PCNA (sc-56, Santa Cruz) as the loading controls.

Anti-DBN M2F6 (1:1000 for western blot, 1:100 for immunohistochemistry, ADI-NBA-110-E Enzo), anti-ATM 2C1(1A1) (1:200, ab78 Abcam), anti-pS1981-ATM 10H11.E12 (1:400, 200-301-400S, Rockland), anti-p-p44-42 ERK (pMAPK, 1:1000 #9101, CST), anti-α-tubulin (1:5000, T6199, Sigma), anti-GAPDH (1:5000, CB1001, Merck), anti-pS15-p53 (1:1000 #9284, CST), anti-poly-monoubiquitin FK2 (1:1000, BML-PW8810, Enzo), anti-HA 3F10 (1:1000, 11867423001, Roche), anti-FLAG M2 (1:10,000, F3165, Sigma), anti-cyclin B1 (1:1000, sc-245, Santa Cruz), anti-MAP2 mouse (1:500 for immunocytochemistry M9942, Sigma), anti-MAP2 guinea pig (1:1000 for FUNCAT-PLA immunocytochemistry or 1:500 for immunohistochemistry, 188 004 Synaptic System), anti-CaMKII (1:250, ab92332 Abcam), anti-pS647-DBN (1:5000 for western blot, 1:250 for immunohistochemistry, numbering of amino acids refers to the largest human DBN isoform (DBN1 iso3^{22 (link)}), anti-pS142-DBN^{17 (link)} (1:500), anti-DBN-1 C. elegans specific (1:500)^{32 (link)}, anti-biotin mouse (1:5000, B7653 Sigma) or anti-biotin rabbit (1 :5000, #5597 Cell Signalling). The most important uncropped western blots are included in the Supplementary information.

Constructs of HA-tagged CDC20 and CDH1, lentivirus plasmid containing shRNA targeting human CDC20 and CDH1, and his-tagged ubiquitin were generous gifts from Prof. Jinfang Zhang (Medical Research Institute of Wuhan University). Constructs of Flag-tagged wild-type GSDME, shRNA targeting mouse Gsdme and Cdc20, the N-terminal of CDC20, and WD40 domains of CDC20 were purchased from MiaoLingPlasmid. GSDME deletion degron (RNFL) mutant was generated using the QuikChange II XL Site-Directed Mutagenesis Kit (200521, Stratagene). The antibodies used in immunoblot were listed as follows: mouse monoclonal antibodies against CyclinB1 (sc-245, Santa Cruz, USA), CDC20 (sc-13162, Santa Cruz, USA), CDH1/FZR1 (sc-56312, Santa Cruz, USA), Vinculin (MAB6896, R&D, USA), HA-tag (66006-2-Ig, Proteintech, USA), Flag-tag (66008-4-Ig, Proteintech, USA); rabbit monoclonal antibody against GSDME (ab215191, Abcam); rabbit polyclonal antibody against GSDMD (96458, Cell Signaling Technology, USA), Caspase-3 (9662, Cell Signaling Technology, USA), Cleaved Caspase-3 (9664, 5A1E, Cell Signaling Technology, USA), Cdc20 (10252-1-AP, Proteintech, USA), Actin (AF5003, Beyotime, China), HA-tag (51064-2-AP, Proteintech, USA), Flag-tag (20543-1-AP, Proteintech, USA).