Huh 7.5.1 cells were infected with HCV or dengue virus at 0.1 MOI. After 3 days, the culture medium was replaced with exosome-depleted FBS medium. After 24 h, (A) extracellular ATP levels were measured using a luminescent cell viability assay and (B) intracellular calcium levels were measured using a fluorometric calcium assay. (C) Neutral sphingomyelinase (nSMase) activity and (D) protein levels of Panx1, P2X4, activated caspase-3, and GAPDH were measured from cell lysates. Cells were stained with TO-PRO-3 and annexin V and determined by flow cytometry. (E) TO-PRO-3 uptake was measured in TO-PRO-3 low and intermediate/Annexin V low and high cells. (F) Annexin− early apoptotic cells were identified by forward scatter (FSC) intermediate and high/TO-PRO-3 high in side scatter (SSC) intermediates and high/annexin V low cells. Data are expressed as mean ± SD (n=3). *P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant, as assessed using an unpaired student t-test.