Figure 1.

Effect of HIV-1 Nef expression on autophagy in Primary Human Astrocytes (PHFA). (A) Immunocytochemistry. PHFA cells were plated in 2 well chamber slides in astrocyte growth medium. Adenovirus transductions and fixing the cells were performed as described in the Materials and Methods. The fixed cells were probed with anti-GFAP antibody for astrocyte marker and anti-HIV-1 Nef (SF2) then mounted on slides using DAPI solution. The slides were visualized using a fluorescence microscopy. Green is GFAP; Red is HIV-1 Nef and blue is DAPI. (B) Immunoblotting of Cell lysates obtained from PHFA transduced with Ad-Nef or Ad-Null. PHFA cells were harvested and lysed using TNN buffer containing 1% NP40 supplemented with mammalian protease inhibitors at 4°C followed by SDS-PAGE and Western blots as described in Materials and Methods. Three different cultures of PHFA were used. The results are representative of at least 3 independent experiments. (C) MTT Assay. MTT proliferation assay was performed as described in Materials and Methods. The experiments were performed in duplicates in a 96 well plate and averages are shown as bar graphs. MTT activity is described differences in absorbances between 620 nm and 595 nm. (D) PHFA cells were transduced with Adnull and AdNef, harvested and lysed at 10, 24, and 48 hrs post-transductions (hpt) using TNN buffer containing 1% NP40 supplemented with mammalian protease inhibitors at 40C followed by SDS-PAGE and Western Blots as described in Materials and Methods.