Figure 5

PERK-dependent ROS production has a positive effect on autophagy induction at later stages of dengue (Den) infection. (a) In MDCK cells, the inducer of ROS pyocyanin (100 μM) gradually increases ROS (h post treatment (HPT)). ROS scavenger NAC (5 mM) partially blocks the increase in ROS. (b) NAC decreases dengue-induced autophagy (LC3-II, 16 kDa) at later stage (48 HPI). (c) Although there is little change in ROS during the first 24 HPI, by 48 HPI dengue (Den) induces an ∼30% increase in ROS (48 HPI compare Den with Mock). NAC prevents this increase, as does salubrinal (Sal) or the combination of the two. The commonly used inhibitor of autophagy wortmannin (Wort; 100 nM) and the ATM inhibitor KU55933 (ATMi) had no effect, and NAC was equally effective in their presence. ****P<0.001 for the bracketed comparisons. (d) The percent changes in ROS levels, compared with mock, in different samples (infected/treated) are presented in this table. NAC (a reducer of ROS) and Sal (an inhibitor of the PERK pathway) suppress the increase in ROS induced by dengue but neither ATMi nor Wortmannin (ATM and phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway inhibitors respectively) have much impact either with or without NAC. We conclude that induction of ROS by the virus does not follow the ATM or the PI3K pathways