Cells and virus
The porcine placental trophoblast cells (PTCs) were isolated from healthy gilts and immortalized by telomerase reconstitution as described in our previous study [11]. The PTCs were cultured in medium 199 (11150059, Gibco, USA) supplemented with 10% FBS (FSP500, ExCell Bio, China), 100 U/mL penicillin and 100 µg/mL streptomycin (P1400, Solarbio, China), at 37°C in a 5% CO2 incubator. The PPV XY strain (Genbank: MK993540) was propagated in porcine kidney-15 (PK15) cells, and the titer was 106.12 TCID50/0.1 mL. Fibrinogen (F3879, Sigma-Aldrich, USA) was dissolved in a 0.9% sodium chloride solution to achieve a final concentration of 10 mg/mL, the fibrinogen solution was then mixed with PPV in a volume ratio of 9:1 and loaded into a transparent bag for UV irradiation, when the irradiation dose reached 900 J/m2, the titer of PPV virus decreased by 4 log s, resulting in the generation of UV-inactivated PPV. Viral titers were determined as 50% tissue culture infectious dose (TCID50) measurement in PTCs according to the Reed-Muench method.
Inhibitors and antibodies
The apoptosis inhibitor, Ac-DEVD-CHO (C1206), was purchased from Beyotime Biotech (Shanghai, China). Mouse anti-VP2 monoclonal antibody (bsm-41390M) was purchased from Bioss (Beijing, China). Mouse anti-ZBP1 monoclonal antibody (sc-271483) and mouse anti-RIPK3 monoclonal antibody (sc-374639) were purchased from Santa Cruz (USA). Rabbit anti-p-RIPK3 monoclonal antibody (93654S) was purchased from CST (Danvers, MA, USA). Mouse anti-Caspase-8 monoclonal antibody (ab32397) was purchased from Abcam (England). Rabbit anti-MLKL polyclonal antibody (21066-1-AP) was purchased from Proteintech (Wuhan, China). Rabbit anti-p-MLKL monoclonal antibody (AP1244) was purchased from ABclonal (Wuhan, China). Mouse anti-β-actin monoclonal antibody (13C000500) was purchased from GenScript (Nanjing, China).
Transmission electron microscopy
Porcine PTCs were mock-infected or infected with PPV at a multiplicity of infection (MOI) of 1 for 72 h, after which the cells were washed with PBS and fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde and 4% paraformaldehyde for 5 h at 4℃. The cells were harvested and washed with PBS, followed by post-fixation in 2% osmium tetroxide and dehydration by serial washing in 50%, 70%, 90%, 95%, and 100% ethanol. The tissues were embedded, cut into thin sections, and viewed with a transmission electron microscope (FEI Inc.).
Lactate dehydrogenase cytotoxicity assay
The cells were mock-infected or infected with PPV at a MOI of 1 for the indicated time in the presence of the apoptosis inhibitor, Ac-DEVD-CHO, and then the release of lactate dehydrogenase (LDH) was measured using an LDH cytotoxicity assay kit (C0016, Beyotime Biotech) according to the instructions.
Western blot
The cells were lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail. Equal amounts of protein from each extract were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with PBS containing 5% skim milk and 0.1% Tween-20 for 1 h at room temperature, the membrane was incubated with primary antibody overnight at 4°C, washed, and then incubated with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature. The membrane was incubated with enhanced chemiluminescence (ECL) reagents in the dark, imaged with a photodocumentation system, the primary protein bands were identified, and their intensity was quantitated with Image J software.
Immunofluorescence assay of MLKL by confocal microscopy
To examine the cellular localization of MLKL, cultured porcine PTCs were mock-infected or infected with PPV at a MOI of 1 for 72 h, then washed with PBS and fixed with ice-cold 4% (wt/vol) paraformaldehyde for 20 min at room temperature, followed by incubation with 0.1% Triton X-100 for 20 min. The cells were incubated with anti-MLKL Abs, followed by the corresponding fluor-conjugated secondary Ab. The nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI) and fluorescence was observed with a laser scanning confocal microscope (Leica, TCS SP8).
ZBP1 mRNA expression levels by real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from PTCs with TRIzol, verified by 260/280 ratio, and reverse-transcribed into cDNA using a kit. The relative levels were determined by RT-qPCR using the following primers: ZBP1-F: ATGGCAACGAGATGAGACT; ZBP1-R: AGGAAGCACGAGCGAATT.
Silencing of ZBP1 in PTCs by transfection with siRNA
The ZBP1-specific siRNA was synthesized by Synon Biotechnology Co., Ltd, (China) and the sequences were 5'-CACCAGCAAAGAAACACCUTT-3' (siZBP1-638), 5'-GCAACUGUUAGAGAAAGCATT-3' (siZBP1-759), and 5'-GUCUGAATCCACGAUUGGAGT-3' (siZBP1-1029). The porcine PTCs were seeded in 6-well or 96-well plates at a density of 2×105 cells/well or 2×104 cells/well, respectively. After adhesion, the cells were transfected with 40 pmol of ZBP1 siRNA per well in a 6-well plates and 4 pmol of ZBP1 siRNA per well in a 96-well plates using Lipofectamine 6000 transfection reagent (C0526, Beyotime Biotechnology, China) according to the manufacturer's instructions.
Generation of ZBP1, Caspase-8, RIPK3, and MLKL knockout PTCs
Three pairs of guide RNAs (gRNAs) were designed for each porcine gene: ZBP1, Caspase-8, RIPK3, and MLKL and are shown in Table S1. The gene knockout identification primers used are listed in Table S2. The synthesized gRNAs were phosphorylated and subcloned into the lentiCRISPRv2 vector (#52961). The recombinant plasmid and the packaging plasmids, psPAX2 and pMD2.G, were co-transfected into HEK293T cells, and the recombinant lentiviruses were collected 72 h after transfection. The PTCs were infected with the recombinant lentiviruses for 48 h, and then selected with puromycin. Positive cells were obtained and transferred to 96-well plates for monoclonal growth.
Statistical analysis
All experiments were independently performed three times, and the results are representative of two or three independent experiments. Data are expressed as mean ± SD and analyzed by ANOVA or unpaired Student’s t tests. Statistical significance was defined as p < 0.05.