FIG. 5.

ROS directly activated MAPK and PI3K/Akt signaling pathways in A. stephensi cells in vitro. Exogenous H₂O₂ induced MEK, ERK, and p38 phosphorylation in A. stephensi ASE cells (A, B) and MSQ43 cells (C, D). For catalase treatment, cells were pretreated with 200 units/ml of catalase for 40 min before treatment with increased doses of H₂O₂. Blots are representative of three independent experiments. Fold changes of pMEK, pERK, and p-p38 treated with 100 and 500 μM H₂O₂ in ASE cells (B) and 100 μM H₂O₂ in MSQ43 cells (D) are represented relative to paired PBS controls. Pairwise comparisons of treatments were analyzed by Student's t-test (α = 0.05). Data are represented as means ± SEMs from three independent experiments and significant differences within treatment pairs are indicated. (E) Increasing doses of H₂O₂ activated FOXO and p70S6K phosphorylation in ASE cells and pretreatment with catalase at 200 units/ml for 40 min before H₂O₂ treatment reduced this phosphorylation. Blots are representative of three independent experiments. Fold changes of pFOXO and p-p70S6K are shown in Supplementary Figure S3. (F) NOS gene expression was analyzed in ASE cells by quantitative reverse transcriptase–polymerase chain reaction at 24 h post-treatment. Fold inductions of NOS relative to paired controls from three independent experiments, represented as means ± SEMs, were analyzed by Student's t-test (α = 0.05). No significant differences within treatment pairs were observed. NOS, nitric oxide synthase; H₂O₂, hydrogen peroxide.