Journal: Journal of Virology
Article Title: Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication
doi: 10.1128/jvi.00064-24
Figure Lengend Snippet: Downregulation of ceramide levels promotes PoRV replication. (A) Schematic representation of ceramide formation pathways and respective inhibitors. (B and F) IPEC-J2 cells were treated with the inhibitors at the indicated concentrations for 24 h. CCK8 assay was used to evaluate their cytotoxicity on IPEC-J2 cells. (C and G) IPEC-J2 cells were treated with the inhibitors for 12 h; then, all cells were collected for total lipid extraction. LC-MS analysis was applied to measure the abundance of C24 in cells compared with in untreated cells. (D–E and H–I) IPEC-J2 cells pretreated with Fumonisin B (50 µM), Myriocin (20 µM), GW4869 (10 µM), or Amitriptyline (30 µM) for 12 h and then infected with PoRV (0.1 MOI) for 6 h. Cells and supernatants were harvested for analysis of the relative levels of PoRV NSP5 mRNA (D and H) and viral titers, respectively (E and I). The data were generated from three independent experiments (means ± SD). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Article Snippet: Myriocin [a potent inhibitor of serine palmitoyltransferase (SPT) served as the rate-limiting enzyme in the de novo pathway], Fumonisin B1 (an inhibitor of ceramide synthases in both the de novo and salvage pathways), Amitriptyline HCl [an inhibitor of the acid sphingomyelinase (SMase)], GW4869 (an inhibitor of the neutral SMase), and Z-VAD-FMK (an inhibitor of apoptosis) were obtained from GLPBIO (Montclair, CA, USA) and dissolved in sterile dimethyl sulfoxide at concentrations ranging from 5 mM to 20 mM ( 43 , – 46 ).
Techniques: CCK-8 Assay, Extraction, Liquid Chromatography with Mass Spectroscopy, Infection, Generated
