Human T-cell lines A3.01 and Jurkat (a clone with high expression of CD4), Luminespib mouse ACH-2 cells harboring an integrated HIV-1 provirus (clone #4; Clouse et al., 1989), and A2 and H12 clones of Jurkat cells latently infected with a ‘‘mini-virus’’ containing the HIV-1 LTR-Tat-IRES-EGFP-LTR (Blazkova et al., 2009 and Jordan et al., 2003) were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 12.5 mM Hepes, and antibiotics

(penicillin 1 × 105 U/l, streptomycin 100 mg/l; 10% FBS-RPMI). The cells were treated with increasing concentrations of HA (1.25 and 2.5 μl/ml of HA correspond to 31.25 and 62.5 μg/ml of hemin or 48 and 96 μM hemin, respectively). ACH-2, A2 and H12 cells were stimulated with phorbol myristate acetate (PMA; final concentration 0.5 ng/ml was used throughout the experiments) to express HIV-1 or EGFP, respectively. The cells were also treated with N-acetyl cysteine (final concentration

5 and 10 mM), SnPP (final concentration 6.25 μM), TNF-α (final concentration 1 and 10 U/ml), PHA (final concentration 0.5 and 1 μg/ml). The stock of HIV-1 was prepared using a transient transfection of Jurkat cells with pNL4–3 (Adachi et al., 1986). The culture supernatant was collected at day 7 after transfection and virus titer was estimated as 4.8 × 1010 TU/ml (transducing units/ml) based on levels of p24 antigen determined by RETRO-TEK HIV-1 p24 antigen ELISA according to the manufacturer’s protocol. For time course experiments, 0.2 × 106 cells in 0.2 ml of 10% FBS-RPMI were infected with www.selleckchem.com/products/CP-690550.html 2 μl of the stock; after 4 h of adsorption of inoculum, 0.8 ml of 10% FBS-RPMI was added and supplemented with HA (final concentration 1.25 and 2.5 μl/ml). The cells were split 1:4 at the indicated times after infection and the media was supplemented with HA to keep the final concentrations as indicated. The growth of HIV-1 was characterized by levels of p24 antigen in culture supernatants. For detection of HIV-1 reverse

transcripts, virus stock was treated with RNAse-free DNase I (Sigma, Germany; final concentration 300 U/100 μl of virus stock) and incubated at room temperature for 45 min to remove plasmid and cellular DNA present in the inoculum. 0.5 × 106 A3.01 and Jurkat cells in Erastin price 0.2 ml of 10% FBS-RPMI were infected with 100 μl of the DNase I-treated virus stock, and after 4 h of adsorption of inoculum, 0.8 ml of 10% FBS-RPMI was added and supplemented with HA (final concentration 2.5 μl/ml) or Azidothymidine (AZT; final concentration 10 μM) as a control. Forty eight hours after infection, the cells were collected in PBS, trypsinized and used for DNA isolation. Total cellular DNA was isolated using a modified method of Miller’s salting-out procedure, without proteinase K and with addition of a chloroform extraction phase (Olerup and Zetterquist, 1992).