Tat basic region retrieves sequestered bFGF into a soluble form that mediates Tat-promoted vascular cell growth. (A) shows the mean of bFGF levels (in picograms per milliliter) retrieved from cells (left panel) or ECM (right panel). KS cells were lifted with a cell dissociation buffer, and both the suspended cells or the plates containing the ECM produced by the cells were incubated with Tat (25 ng/mL), [46-60] Tat (4 ng/mL), [56-70] Tat (4 ng/mL), heparin (30 μg/mL), or dilution buffer. A limited trypsin digest of suspended cells or plates was used to retrieve the total bFGF bound to cells or ECM.16 bFGF was then measured in the supernatants by ELISA. (B) shows the mean values of bFGF (in picograms per milliliter) maintained in a soluble form by Tat or Tat basic peptide. KS cells were cultured in the presence of exogenous bFGF (1 ng/mL) and then incubated for 20 minutes in control buffer (PBS-0.1% BSA), Tat (1 or 25 ng/mL), [46-60] Tat (0.2 or 4 ng/mL), heparin (30 μg/mL), or Tat (25 ng/mL) and heparin (30 μg/mL) combined sequentially. (C) The left panel shows proliferative assays with KS cells cultured for 48 hours with 0.5 μmol/L antisense bFGF (ASbFGF) or sense bFGF (SbFGF) oligomers.24 Tat (1 ng/mL) and ³[H]-thymidine were then added to the cells, and growth was monitored after 48 hours. Results (from 3 experiments, in 5 replicates) are expressed as the percentage increase of³[H]-thymidine uptake after the addition of Tat as compared with basal cell growth (0% increase, which was 1,874 ± 20 cpm). In the middle and right panels, anti-bFGF antibodies (10 μg/mL) were added to KS cells before the addition of Tat (1 ng/mL) or [48-53] Tat (0.1 ng/mL). The antibody buffer (PBS-0.1% BSA) was the negative control. Results (from 4 experiments, each in duplicate) were obtained by the cell counting method and expressed as the percentage increase of cell growth over the number of KS cells grown in the absence of mitogens (basal cell growth), which was 1 ± 10⁴ cells/well. (D) shows proliferative assays with IC-HUVE cells induced to proliferate with 1 ng/mL of Tat or 20 ng/mL of aFGF, in the presence or absence of anti-bFGF antibodies (10 μg/mL). (E) shows HUVE cell growth experiments with bFGF or VEGF (5 ng/mL each) in the presence or absence of 1 ng/mL of Tat. For (D) and (E), results from 4 experiments performed by the cell counting method refer to the number of cells collected 4 to 5 days after the addition of bFGF, aFGF, VEGF, or Tat. They are expressed as a percentage increase of cell growth over the number of cells grown in the absence of mitogens (basal cell growth).