Fig. 1
Characterization of HIV-1 chronically infected U937-based cell lines expressing Nef in a regulatable way. a Determination of viral release from HIV-1 chronically infected U937-based cell lines by HIV-1 CAp24 ELISA. Cultures of 106 cells/mL of U937 cells either uninfected or chronically infected with Δnef HIV-1, the latter stably transfected with vectors expressing either ER alone (U937 Δnef HIV-1), ER fused with wt Nef (U937 Δnef HIV-1/wt Nef), or ER fused with the Nef G2A mutant (U937 Δnef HIV-1/NefG2A), were carried out for 48 h with either HT or equal volume of vehicle. Afterwards, supernatants were harvested, clarified, and viral contents measured in terms of concentration of CAp24. Shown are the mean values +SD as calculated from duplicate conditions run in seven independent experiments. Nd: not detectable. b Western blot analysis for expression of HIV-1 related products in the HIV-1 chronically infected U937 cell lines either untreated (−) or treated (+) with HT for 48 h. Cell lysates from the U937-based cell lines were resolved in 12 % SDS-PAGE and probed for Gag, Env (upper panel), and Nef expression (middle panel). Signals were normalized by β-actin detection (lower panel). On the right of each panel, molecular weight markers are given in kilodaltons (kDa). On the left, migrations of relevant products are indicated. The results are representative of five independent experiments. c FACS analysis for the expression of both Gag and Nef products. The different U937-based cell lines were incubated for 48 h with HT or equal volume of vehicle, then permeabilized and labeled with both anti-Gag and—Nef mAbs. Percentages of events are reported in the respective quadrants. The results are representative of two independent experiments