Fig. 6

Influence of calphostin C and H88 on HIV-1 LTR-driven reporter gene expression in PMA or TNF-α-treated Jurkat cells

Jurkat cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum under standard tissue culture conditions described above. The cells were transfected with 2.5 µg of pHIV-1 LTR-CAT (12) by the DEAE-dextran procedure (47). Sixty-eight hours after transfection the cells were treated with calphostin C (A and D, under light) or H88 (B and E) at the indicated concentrations and incubated in CO₂ (5%) incubator at 37°C for .5 hr. Cells were then treated with PMA (20 ng/ml; A, B, and C) or TNF-α (10 ng /ml; D, E, and F) and incubated for an additional 3.5 hr at 37°C in CO₂ (5%) incubator. The cells were then harvested, extracts were prepared, and CAT activity in 10 µg (protein) of cell extracts was determined as described previously (3). The (C) and (F) show percent inhibition of CAT activity at the indicated concentrations of the drugs in PMA-stimulated (C) or TNF-α (F)-stimulated cells. The quantitation of CAT activity was made by densitometric scanning of the autoradiographic signals. CAT activity is expressed as percentage ¹⁴C-chloramphenicol (CAP) derivatives produced per hour in the presence of 10 µg of protein in the cell extract. CAT activity in the absence of the drug is considered as 0% inhibition.