PKC-delta is required for HIV-1 BaL replication in human macrophages. (A) Macrophages (5 × 105) were pretreated for 30 min with Ro31-8220, an inhibitor of all PKCs, or by selective inhibitors of certain PKC isozymes (rottlerin, PKC-delta inhibitor, Hispidin PKC-beta inhibitor and Gö6976, PKC-alpha, betaI, and mu inhibitor) at indicated concentrations. HIV-1 BaL infection (1 ng p24/ml) lasted 3 h. After 3 days culture supernatants were collected and viral replication assessed by the presence of p24 determined by ELISA. (B) 5x105 macrophages were preincubated with rottlerin for 24 h and then cultured in rottlerin free medium for 24 h before infection to evaluate cytotoxic effects of rottlerin (rottlerin + wash). As positive control, the total inhibitory effect of rottlerin was evaluated as described in panel A. Error bars represent 3 experiments with one donor. Results are representative of experiments achieved in three different donors. (C) HeLa LTR-beta-gal cells or (D) macrophages were preincubated with siRNA against PKC-delta or scrambled siRNA for 2 days. Cells were then infected with HIV-1 BaL (1 ng p24) for 3 h and washed. After 48 h, cells were treated with X-gal and infection was assessed by counting beta-gal positive blue cells using microscopy (C) or by quantifying p24 in the supernatant (D). Western blot with anti PKC-delta antibodies is shown as the control. (E) Macrophages (5x106) were infected with HIV-1 BaL (1 ng p24/ml) or stimulated by PMA for 30 minutes, and the activation of PKC isozymes was determined by analyzing PKC translocation to the membrane. Membrane bound (M) and cytoplasmic (C) proteins were extracted and separated by 10% SDS-PAGE. PKC-delta and betaII isozymes were visualized by chemiluminescence using specific antibodies for each isozyme. Homogeneity of protein extracts was controlled by amido black staining of membranes. The purity of membrane and cytoplasmic fractions was controlled by Western blotting for SLC44A1 (membrane) and Hsp70 (cytoplasm). Results are representative of three independent experiments.