Figure 5.

gp120-induced NF-κB is essential for the production of TNF-α and IL-10. (A) monocytes (3 × 10⁶) were stimulated by X4-gp120 during 5 to 60 minutes. Total cell proteins lysate were separated by SDS-PAGE and analyzed by Western blotting for IκB degradation. Images were processed by slight contrast adjustment using Image Lab 5.2.1 software and cropped to focus on protein of interest. (B) HeLa cells (4 × 10⁴ cells) stably transfected with a plasmid coding for β-galactosidase gene under the control NF-κB inducible promotor were co-cultured with the same amount of HeLa cells expressing X4-gp120 of HIV-1 Lai isolate. After 20 hours of co-culture, β-galactosidase activity was evaluated by adding the substrate of X-gal. Blue cells corresponding to β-galactosidase positive cells were quantified by scoring under light microscopy. The inset figure showed the nuclear translocation of NF-κB P65 subunit after 30 and 120 minutes of co-culture as described above. (C) Monocytes were incubated with HIV-1 gp120 (10 nM) in presence or absence of NF-κB inhibitors Bay117082 or TLCK. After 24 hours, cell supernatants were harvested and quantified by ELISA for the presence of TNF-α and IL-10. Statistical significances were compared to gp120 treated cells in the absence of inhibitor.