Fig. 4.

Effect of imiquimod on the PKA pathway. (A) Immunoblot images of p-CREB and CREB expression, and quantitative densitometric analysis in HEp-2 and A549 epithelial cells after exposure to either vehicle or imiquimod (10 μg/ml) during the indicated times. (B) Immunoblot images of p-CREB and CREB expression, and quantitative densitometric analysis in HEp-2 and A549 cells after exposure or not to R-PIA (10 μM) or KT5720 (10 μM) pretreatment for 15 min, and then stimulated or not with imiquimod (10 μg/ml) for 10 min. (C) HEp-2 and A549 cells were treated or not with R-PIA (10 μM) or KT5720 (10 μM) for 15 min, and then infected with RSV A2 (moi = 1) and treated or not with imiquimod plus R-PIA or KT5720 for 24 h. Total virus yields were determined by plaque assay in Vero cells and plotted as the percentage of inhibition with respect to untreated–infected control (CV). (D) Expression of cystatin A in HEp-2 and A549 cells treated with imiquimod (10 μg/ml). At the indicated times after treatment, total RNA was extracted and gene expression was determined by real-time PCR. Data were analyzed using the 2^−ΔΔCt formula. Actin was used as an internal for determination of gene expression. (CC): cell control (unstimulated and uninfected cells). (CV): untreated–infected control cells. Data represent mean ± SD for n = 3 independent experiments, performed in duplicate. *Significantly different from CV (p-value <0.05); ^# significantly different from CC (p-value <0.05); ^¥Significantly different from imiquimod treated cells (p-value <0.05); One way ANOVA with Tukey post test. There were no statistically significant differences between imiquimod treated cells and imiquimod plus R-PIA treated cells regarding both p-CREB expression and virus yields.