Figure 6  IFNα treatment inhibits HIV-1 replication and disrupts IL-1β and miR-146a expression. CD4+ T-cells were infected with HIV-1 alone, infected with HIV-1 and treated with Meth daily, or infected with HIV-1 and treated with IFNα daily. (A) Culture supernatants were analyzed for HIV-1 replication by p24 ELISA, and p-values were calculated relative to HIV+ controls (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Cells were harvested at 2 days P.I. miR-146a and IL-1β mRNA expression was analyzed by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IFNα to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to HIV+ controls (**p < 0.01). (C) Culture supernatants were collected 2 days P.I. and analyzed for IL-1β concentration by ELISA. (*p < 0.05, ***p < 0.001). (D) Cells harvested at 2 days P.I. were analyzed for TRAF6 and IRAK1 mRNA expression by RT-qPCR. Fold change was calculated by normalizing HIV+Meth or HIV+IFNα to HIV+ samples. Data represent the mean ± SD of 3 independent experiments, and p-values were calculated relative to HIV+ controls (***p < 0.001). (E) Protein extracts from cells harvested at 2 days P.I. were analyzed for TRAF6 and IRAK1 expression by Western Blot analysis. GAPDH was used as a loading control. Relative band intensity was calculated using ImageJ software, and p-values were calculated relative to HIV+ controls (***p < 0.001).