Cell lines and culture conditions

The human hepatoblastoma cell line HepG2 and hepatocellular carcinoma Hep3B [18] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Beijing Biyun Tian Biological Technology Co., Ltd., China). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

Xenograft tumorigenesis model

Six-week-old female nude mice (Nu/Nu strain) were obtained from Beijing Vital River Laboratory Animal Technology Limited Company (Beijing, China) and randomly assigned to five groups. A total of 1 × 106 HepG2 cells were subcutaneously injected into each mouse, and the tumor volume was measured every 2 d. After 16 days of drug treatment, the mice were euthanized, and the xenograft tumors were collected. Tumor volumes were determined with the following standard formula: length × width2/2. All animal experiments were carried out with the endorsement of the Institutional Animal Care and Use Committee of Harbin Medical University (approval number: IRB3014822) and adhered to the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals.

Cell Counting Kit-8 assay

The Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell viability. HepG2 and Hep3B cells were seeded at a density of 5 × 103 cells per well in a 96-well plate. Following drug, plasmid or siRNA treatment, each well of the 96-well plate was supplemented with 10 μL of CCK-8 reagent and 100 μL of DMEM medium. The plate was then incubated for 1 h. Subsequently, the absorbance values were measured at 450 nm using a Power Wave HT microplate spectrophotometer (Bio Tek, USA). The reagent Z-VAD-FMK (#HY-16658B), necrostatin-1 (#HY-15760) and disulfiram (#HY-B0240) were purchased from MedChemExpress and used at concentrations of 20 µM, 10 µM and 1 µM, respectively.

Colony formation assay

The cells were seeded into 6-well plates at a density of 500 cells per well. The cells were cultured at 37 °C, 5% CO2 incubator for 14 days to allow colony formation. The cells were subsequently fixed with 4% formaldehyde for 30 min, washed twice with PBS, and stained with 0.1% crystal violet for 20 min after air drying. Following another two washes with PBS, the colonies were photographed and counted.

5-Ethynyl-2’-deoxyuridine (EdU) staining assay

A Cell-LightTM EdU Apollo In Vitro Kit (RiboBio, Guangzhou, China) was used to assess cell proliferation. The cells were seeded in 24-well plates (NEST, Hong Kong, China) at a density of 2 × 104 cells/well. After treatment, the cells were incubated with 50 μM EdU at 37 °C for 120 min. Following permeabilization with 0.5% Triton X-100, the Apollo staining solution was added to the cell culture medium and incubated in the dark for 30 min. Finally, the cells were incubated with 20 μg/mL Hoechst 33342 for 30 min. The EdU index (%) was calculated as the average ratio of the number of EdU-positive cells to the total number of cells with a confocal laser scanning microscope.

Flow cytometry

An Annexin V-AbFluorTM 488/PI Apoptosis Detection Kit (Abbkine, Wuhan, China) was used to assess apoptosis following the manufacturer’s instructions. The cells were rinsed with precooled PBS twice and then centrifuged at 1500 rpm for 5 min. The cells were resuspended in 400 μL of 1× binding buffer, and 5 μL of FITC-labeled Annexin V and 2 μL of propidium iodide (PI) were added to the cell suspension for staining, which was carried out for 15 min. Data analysis was performed with CytExpert software.

Cellular thermal shift assay

The cellular thermal shift assay (CETSA) was conducted following previously described methods [19]. The cells were treated with either DMSO or KLX for 1 h. Subsequently, the cells were lysed in 100 µL RIPA buffer, and the lysate was divided into equal-volume aliquots. Each aliquot was subjected to heat treatment at a specified temperature for 5 min. After heating, protein immunoblotting was performed with established protocols as previously described.

Western blot

Proteins were extracted from cells with RIPA buffer. Concentrations were determined with a BCA assay (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes. Primary antibodies against ZBP1 (1:1000; #A13899, ABclonal, China), ZBP1 (1:1000; #13285-1-AP, Proteintech, China), AIM2 (1:1000; #A3356, ABclonal, China), RIPK1 (1:1000; #A7414, ABclonal, China), Bcl-2 (1:1000; #A19693, ABclonal, China), Bax (1:1000; #60267-1, Proteintech, China), NLRP3 (1:1000; #A12694, ABclonal, China), N-GSDMD (1:1000; #AF4012-100, Affinity, USA), RIPK3 (1:1000; #17563-1-AP, Proteintech, China), p-RIPK3 (1:500; #AP1260, ABclonal, China), MLKL (1:1000; #A19685, ABclonal, China), p-MLKL (1:1000; #AP0949, ABclonal, China), IL-1β (1:500; #WL02257, Wanleibio, China), Caspase-8 p18 (1:1000; #WL00659, Wanleibio, China), HOXD10 (1:500; #TA800777S, OriGene, USA), RNF180 (1:1000; #orb541388, Biorbyt, UK), ubiquitin (1:1000; #10201-2-AP, Proteintech), and GAPDH (1:1000; #TA-08, ZSGBbio, China) were incubated overnight at 4 °C followed by secondary antibodies (1:10000; LI-COR Bioscience, Lincoln, NE, USA) for 50 min. Blots were scanned with an infrared fluorescence imaging detector (LI-COR Odyssey).

DARTS

HepG2 cells were treated with KLX for 1 h, followed by three washes with the PBS. The cells were lysed with lysis buffer containing protease inhibitors (Roche, Switzerland) and collected after treatment on ice. The supernatant was collected by centrifugation at 12000 rpm for 10 min at 4 °C. After TNC (500 µM Tris-HCl, 500 mM NaCl, 100 mM CaCl2, pH 8.0) was added at a 1:10 ratio, the protein concentration was determined with a BCA protein kit (Beyotime Institute of Biotechnology, Shanghai, China), and the supernatant was divided into 3 portions. Different proportions of protease were added as needed. After heating, Western blotting was performed as described previously.

Coimmunoprecipitation (Co-IP)

The cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF, and protease inhibitor). The lysates were cleared by centrifugation at 12,000 × g for 15 min at 4 °C. The supernatant was incubated with 2 µg of primary antibody against ZBP1 (1:1000; #A13899, ABclonal, China) overnight at 4 °C with gentle agitation. Protein A/G agarose beads were then added and the mixture was incubated for an additional 2 h. Beads were washed five times with lysis buffer, and bound proteins were eluted by boiling in SDS sample buffer. The eluates were analyzed by SDS-PAGE and Western blotting with specific antibodies.

Transwell assay

HepG2 and Hep3B cells were seeded at a density of 5 × 103 cells per well in 24-well Transwell chambers. After 8 h, the cells were treated and incubated. After treatment, 24-well plates were filled with paraformaldehyde fixative (Biosharp, Beijing, China), and the chambers were immersed in the fixative for 30 min. Then the chambers were washed with PBS. Clean wells of a 24-well plate were then filled with crystal violet staining solution (Biosharp, Beijing, China), and the chambers were placed in the staining solution for 30 min. Cell migration was captured by imaging software, and the average number of migrated cells was calculated for quantitative analysis.

RNA isolation and quantitative real-time PCR

Total RNA was extracted from HepG2 cells with TRIzol reagent (Invitrogen, Karlsbad, CA, USA) according to the manufacturer’s instructions. A reverse transcription kit (Toyobo, Osaka, Japan) was used to reverse transcribe 1 μg of RNA into complementary DNA (cDNA) at 37 °C for 15 min, 98 °C for 5 min, and 4 °C indefinitely. Real-time quantitative PCR was performed on a 7500 FAST Real-Time PCR System (ABI, Waltham, MA, USA) with SYBR Green (Toyobo). GAPDH was used as an internal reference to quantify messenger RNA (mRNA) levels. mRNA expression was analyzed according to the 2−ΔΔCt method.

ZBP1 (forward primer: 5ʹ-AACATGCAGCTACAATTCCAGA-3ʹ; reverse primer: 5ʹ- AGTCTCGGTTCACATCTTTTGC-3ʹ);

AIM2 (forward primer: 5ʹ-TGGCAAAACGTCTTCAGGAGG-3ʹ; reverse primer: 5ʹ- AGCTTGACTTAGTGGCTTTGG-3ʹ);

RIPK1 (forward primer: 5ʹ-GGGAAGGTGTCTCTGTGTTTC-3ʹ; reverse primer: 5ʹ- CCTCGTTGTGCTCAATGCAG-3ʹ);

GAPDH (forward primer: 5ʹ-AAGAAGGTGGTGAAGCAGGC-3ʹ; reverse primer: 5ʹ-TCCACCACCCTGTTGCTGTA-3ʹ);

18S (forward primer: 5ʹ- CCTGGATACCGCAGCTAGGA-3ʹ; reverse primer: 5ʹ- GCGGCGCAATACGAATGCCCC-3ʹ);

HOXD10 (forward primer: 5ʹ- GACATGGGGACCTATGGAATGC-3ʹ; reverse primer: 5ʹ-CGGATCTGTCCAACTGTCTACT-3ʹ).

Wound healing assay

The cells were seeded in 6-well plates. When the cells reached 90% confluence, a sterile 2.5 μL pipette tip was used to slide straight through the cell monolayer, which was photographed under a microscope. The cells were incubated in serum-free medium, and the wounds were observed and captured. The gap lengths were measured from the photomicrographs.

Luciferase reporter assay

Binding sites between ZBP1 and HOXD10 were identified with bioinformatics tools such as microRNA.org, starBase v2.0, and miRcode. The ZBP1 3’-UTR fragment, which includes either the predicted HOXD10 binding sites or their mutated versions, was synthesized and inserted into the pGL3-basic vector (Promega, Madison, WI, USA). Human HepG2 cells were then cotransfected with this luciferase reporter vector, both in the presence and absence of the ZBP1 mimic. The Dual Luciferase Reporter Assay System (Promega) was used to assess luciferase activity following the manufacturer’s instructions. The results were normalized against Renilla luciferase activity.

Surface plasmon resonance (SPR)

The CM5 chip was inserted into the slot with the labeled side up, and the second channel was activated by the flow of EDC and NHS at 10 μL/min. Ligand protein was immobilized at 50 μg/mL in sodium acetate buffer onto the second channel, followed by blocking with ethanolamine. The first channel served as a reference, with buffer instead of protein. Each sample was diluted to various concentrations in a 96-well plate and allowed to flow over the chip at 30 μL/min for 150 s. The chip was regenerated with 10 mM glycine-HCl (pH 2.0) after each concentration, and the binding constants were determined using Biacore Insight software.

Molecular docking

The crystal structure of ZBP1 was retrieved from the RCSB PDB. Molecular docking was carried out with AutoDock Vina 1.1.2, and visual inspection was performed with PyMol 2.5.4. Interaction energies were predicted through flexible ligand docking simulations within the monomeric pores.

Molecular dynamics

The protein crystal structures of ZBP1, BMI1, and SYVN1 were downloaded from the PDB database [20]. RNF180 was obtained from the AlphaFold database, and the 3D structure of KLX was downloaded from PubChem and energy-minimized with the MMFF94 force field [21]. Protein-protein docking was performed using ZDOCK 3.0.2. KLX and ZBP1 docking was performed with AutoDock Vina 1.1.2, with receptor proteins preprocessed in PyMol and converted to PDBQT format by ADFRsuite 1.04. Energy minimization and molecular dynamics simulations were conducted with AMBER 18, followed by MMGBSA binding free energy calculations based on 90-100 ns MD trajectories.

Statistical analysis

The data are shown as the means ± SDs. Statistical analysis was performed with Student’s t-test for comparing two groups and one-way ANOVA with Tukey’s post hoc test for multiple group comparisons. A P value of less than 0.05 was considered statistically significant. Statistical analysis was conducted with GraphPad Prism 9.