Culture supernatants from HIV-infected PBMCs activate JNK and NF-κB pathway in LX2 cells.

Serum starved LX2 cells were treated with virus-free culture supernatants from HIV-infected (R5- or X4-tropic) human PBMCs for the indicated times. Mock (M) represents control group that was treated with supernatants from uninfected PBMCs. (A) LX2 cell lysates were analyzed for phosphorylation of JNK, Erk and Akt; total levels of these proteins served as the respective loading controls. Data representative of one of three experiments is shown. (B) LX2 cell lysates were analyzed for phospho-c-Jun, c-Fos and phospho-ATF-2 levels; total c-Jun and β-actin served as the loading controls. Data representative of one of three experiments is shown. Values below the lanes show fold-change relative to the mock lanes after normalizing to total c-Jun or β-actin levels. (C) Serum-starved LX2 cells were treated with 25 µM SP600125 (JNK inhibitor) for 2 hr; DMSO was used as control. Culture supernatants from HIV infected PBMCs (mock, R5- or X4-tropic) were diluted 1∶3 with fresh culture medium and added to LX2 cells for 72 hr; the inhibitor was replenished every 24 hr. RNA was isolated from treated LX2 cells and the expression levels of TGF-β and MMP-2 were evaluated by qRT-PCR. Data was normalized using β-actin levels and represents mean ± S.E. of three independent experiments. Values above the bars show p value for comparison of X4/R5/DMSO to either Mock/DMSO or X4/R5/SP6. (D) LX2 cell lysates were analyzed for NF-κB p65 phosphorylation; total levels of p65 served as the loading control. Data representative of one of three experiments is shown. Values below the lanes show fold-change relative to the mock lanes after normalizing to total p65 levels.