HIV-1 Tat-mediated expression of IL-6 and IL-8 involves PI3K/Akt pathway. (a-f) Astrocytes were pretreated with a specific PI3K inhibitor (LY294002) for 1 hour prior to transfection. (a, b) The expression levels of IL-6 and IL-8 at the level of mRNA were determined at 6 hours post transfection by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (c, d) IL-6 and IL-8 protein concentrations in the cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. (e) Astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid for duration of 6 hours and p-Akt levels were measured in whole cell extracts. (f) Astrocytes were transfected for a duration of 6 hours and translocation of p65 was measured. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and LaminB were used as internal loading controls for cytoplasmic and nuclear protein fractions, respectively. A representative Western blot is shown in figures (e) and (f). (g-j) Astrocytes were transfected with either scrambled or Akt1 or Akt2 or Akt3 siRNA for a duration of 48 hours, followed by either mock transfection or transfection with HIV-1 Tat plasmid. (g, h) The levels of IL-6 and IL-8 at mRNA level were determined by real time RT-PCR at 6 hours post transfection. (i, j) The protein concentrations of IL-6 and IL-8 in cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. Statistical analyses was performed by one-way ANOVA and ** denotes P-value of ≤ 0.01 and * denotes P-value of ≤ 0.05.