Reduced DENV-induced TNF-α production and apoptosis in SB203580 treated DENV-infected HepG2 cells and exogenous TNF-α restored the apoptosis. (A) HepG2 cells were infected with DENV serotype 2 at a MOI 5 and incubated 48 h in the presence of either DMSO or 10 μM SB203580. TNF-α mRNA (A) and TNF-α protein (B) expression of DENV-infected HepG2 cells was subsequently examined by real-time RT-PCR and by ELISA, respectively. All data were obtained from three independent experiments and reported as the mean ± SEM. Statistical differences between the groups were tested with an unpaired t-test using StatView version 5.0 and P value less than 0.05 was considered significant. (C) Supernatants containing DENV from mock-infected or DENV-infected HepG2 cells in the presence of either DMSO or 10 μM SB203580 was isolated and incubated with HepG2 cells in the presence or absence of recombinant TNF-α. The percentage of apoptotic cells was then determined at 72 h by annexin V/FITC and PI double staining and quantitation by flow cytometry. Apoptosis cells have annexin V+/PI- staining. (D) Model representing the augmentation of DENV-induced apoptosis mediated by TNF-α via the activation of p38 MAPK and CD137 signaling.