(A) TUNEL labeling of mock- and PEDV-infected cells. proteins and oxidative phosphorylation process were widely involved in the pathological changes and regulation of host cells caused by PEDV contamination, and PI3K/AKT and mTOR signaling pathways played a vital role in antiviral regulation in IPEC-J2 cells. These data might provide Anitrazafen new insights into the specific pathogenesis of PEDV contamination and pave the way for the development of effective therapeutic strategies. for 5?min and the cell pellets were resuspended in PBS containing 5 U RNase and 50?mg/mL of propidium iodide (PI). The cells were then incubated on ice for 30?min in the dark. Cell cycle distribution was calculated from 10,000?cells and determined using a Coulter Epics XL flow cytometer (Beckman Coulter, CA, USA). 2.5. Cell apoptosis assessment by CCK8 Anitrazafen assay and fluorescent staining IPEC-J2 cells were produced in 96-well plates (1??104?cells/well) in 100?L culture medium and infected with CV777 at an MOI of 1 1.0. Control cells were treated with the same dose of culture medium. After washing with PBS, the cells were further cultured in serum-free medium. CCK-8 solution (Beyotime Biotechnology) (10?L) was added to each well at 24, 48, and 72 hpi and Anitrazafen incubated 37?C for 2?h in a cell culture incubator. The absorbance was measured at 450?nm using a 550 Microplate Anitrazafen Reader (Bio-Rad). Experiments were repeated three times, with twelve samples taken at each time point. 2.6. Cell apoptosis detection by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay IPEC-J2 cells were produced on microscope cover slips placed in 6-well tissue culture plates and mock-infected or infected with PEDV at an MOI of 1 1.0.The virus-infected cells were fixed at 48 hpi with 4% paraformaldehyde for 25?min?at 4?C and permeabilized with 0.2% TritonX-100 in PBS at room temperature for 5?min. Cell Anitrazafen samples were rinsed twice with PBS, and the TUNEL reaction mixture (Beyotime Biotechnology) was added and incubated for 60?min?at 37?C, followed by three washes with PBS. TUNEL-labeled cells were subjected to immunofluorescence assay using anti-PEDV N protein mouse monoclonal antibody and goat anti-mouse antibody, as described above. After sealing with an anti-fluorescence quenching liquid, the samples were mounted on a fluorescence microscope and examined at an excitation wavelength of 550?nm and emission wavelength of 570?nm (red fluorescence). 2.7. Cell apoptosis detection by annexin V and PI staining assay IPEC-J2 cells were produced in 6-well tissue culture plates and mock-infected or infected with PEDV at an MOI of 1 1.0.Mock- and PEDV-infected cells were then harvested and washed with cold PBS at 24, 48, and 72 hpi. Cell apoptosis was decided using an AlexaFluor488 AnnexinV/Dead Cell Apoptosis kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The cells were suspended in 100?L annexin-binding buffer, and then incubated with AlexaFluor488-conjugated AnnexinV and PI at room temperature for 15 min in the dark. After incubation, 400?L annexin-binding buffer was added to each DUSP2 sample and mixed gently on ice. The samples were analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter) and Kaluza software. 2.8. RNA-Seq transcriptomic assay IPEC-J2 cells were produced in 25?cm2 cell culture flasks and infected with CV777 at an MOI of 1 1.0. Control cells were treated with the same dose of culture medium. Three replicates were used for each group. Total RNA was extracted from the cells using TRIzol reagent according the manufacturer’s instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKaRa, Japan). cDNA was synthesized using a Super Script double-stranded cDNA synthesis kit (Invitrogen) and sequenced by Shanghai Major bioBiopharm Technology Co. Ltd. (Shanghai, China) using an Illumina HiSeq2500 system (Illumina, CA, USA). Differential expression analysis was carried out using EdgeR software . Differences in expression levels between groups were considered significant after adjusting for multiple testing based on a q value?0.05. We first filtered the genes based on q?0.05 and an absolute difference?>?2-fold (i.e., log2 change?>?1.0). DEG enrichment was analyzed using Gene Ontology (GO) tools (https://github.com/tanghaibao/GOatools), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was carried out using KOBAS (http://kobas.cbi.pku.edu.cn/home.do) . 2.9. Real-time quantitative PCR (qPCR) validation Total RNA was extracted from infected and control IPEC-J2 cells (8 repetitions per group) using TRIzol reagent (Invitrogen) and cDNA was synthesized using a Revert Aid kit (Thermo Scientific, USA) according to the manufacturer’s instructions. Thirteen of the most significantly differentially expressed genes between the PEDV contamination and control groups according to RNA sequencing data (log2-fold change; P?0.05, false discovery rate?>?95%) were selected for real-time qPCR. The primer.