PK-15 (porcine kidney; ATCC CCL-33) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (100 U/ml), and streptomycin (100 mg/ml) (Gibco) at 37 °C under 5% CO2. The FMDV serotype O strain O/CHINA/99 (GenBank accession no. AF506822.2) was maintained by the OIE/National Foot-and-Mouth Disease Reference Laboratory (Lanzhou, China).

Mouse anti-HSP60 (Cat. No. 66041-1-Ig, Dilution: 1:2000), anti-COXIV (Cat. No. 66110-1-Ig, Dilution: 1:2000), and anti-P62 (Cat. No. 66184-1-Ig, Dilution: 1:2000) monoclonal antibodies were purchased from Proteintech (Wuhan, China). A mouse anti-actin monoclonal antibody (Cat. No. CW0096, Dilution: 1:2000) was purchased from CWBIO (Beijing, China). Rabbit anti-Beclin-1 (Cat. No. 3495 S, Dilution: 1:2000), anti-VDAC1 (Cat. No. 4661 S, Dilution: 1:2000), anti-LC3 (Cat. No. 3868 S, Dilution: 1:2000), anti-Drp1 (Cat. No. 8570 S, Dilution: 1:2000), anti-Drp1-Ser616 (Cat. No. 3455 S, Dilution: 1:2000), anti-Parkin (Cat. No. 2132 S, Dilution: 1:2000), anti-CaMKII (Cat. No. 50049 S, Dilution: 1:2000), anti-p-CaMKII (Cat. No. 12716 S, Dilution: 1:2000), anti-ERK2 (Cat. No. 9108 S, Dilution: 1:2000), anti-p-ERK2 (Cat. No. 4370 S, Dilution: 1:2000) monoclonal antibodies were purchased from CST (Danvers, MA, USA). The anti-mouse secondary antibodies conjugated with horseradish peroxidase (HRP) (Cat. No. A0168-1ML, Dilution: 1:2500) and anti-rabbit secondary antibodies conjugated with HRP (Cat. No. 12–348, Dilution: 1:2500) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine RNAi MAX and Lipofectamine 2000 were purchased from Invitrogen (CA, USA). A mitochondria isolation kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

The sequences of target siRNAs (GenePharma, China): 5’-GC-AGAUGCUCGAGCCUUAATT (HSP60); 5’-GUGGCUGUAUGCACAUGAATT (Parkin); 5’-GGGCUAAUGAACAAUAAUATT (Drp1); 5’-CAGCUUGCAUCGCCUAUTT (CaMKII); 5’-GUGCUCUGCUUAUGAU-AAUTT (ERK2); and 5’-UUCUCCGAACGUG-UCACGUTT (NC). Cells were grown to 80% confluence and were transfected with siRNA using Lipofectamine RNAi MAX according to the manufacturer’s instructions. After 6 h, the medium was changed. Incubating cells for 48 h at 37 °C in a CO2 incubator before harvesting.

The cells were collected and lysed with 1 × SDS loading buffer. After SDS-PAGE, the samples were transferred to the NC membrane. The membranes were incubated with 5% skim milk for 1 h at room temperature, followed by overnight incubating with primary antibodies. Next, membranes were washed with TBST. Then, membranes were incubated with a secondary antibody which is conjugated to horseradish peroxidase for 1 h, and TBST was used to wash membranes. The bands on membranes were visualized by incubating with chemiluminescence kit (Thermo Fisher Scientific, USA).

A Mitochondria isolation kit (Cat. No. 89874, Thermo Fisher Scientific, USA) was used in this assay. Firstly, cells were centrifuged at 1000 × g for 5 min. After discarding the supernatant, Reagent A (added with protease inhibitor) was used to resuspend the cells, followed by incubating in ice for 2 min. Next, 10 mL of Reagent B was added to resuspend the cells and further kept on ice for 6 min. Adding 800 mL Reagent C (contains a protease inhibitor). Two times centrifuge (800 × g for 5 min at 4 °C, 15,000 × g for 10 min at 4 °C) were performed. The purified mitochondria were found in the pellet while the supernatant contained cytosolic fraction.

The data presented in this paper are expressed as means and standard deviations (SD) for at least two replicates and were evaluated with a two-way analysis of variance (ANOVA) in GraphPad Prism software version 9 (GraphPad, La Jolla, CA, USA).