Cell–cell fusion
was measured as previously published22 (link) and
using cell lines obtained through the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Target cells were TZM-bl
cells (no. 8129, contributed by J.C. Kappes, X. Wu, and Tranzyme Inc.)
expressing CD4, CCR5, and CXCR4,40 (link) and
containing an integrated reporter gene for firefly luciferase under
control of HIV-1 LTR.41 (link) Effector cells
were HL2/3 (no. 1294, contributed by B. K. Felber and G. N. Pavlakis)
which produce HXB2 Env, Tat, and Rev.42 (link) Serially diluted inhibitors were added to 96-well plates containing
25 000 TZM-bl cells per well cultured overnight. 50 000
HL2/3 cells were added per well, and fusion was allowed to proceed
for 6 h in reduced serum medium (Gibco) with a final concentration
of 1% DMSO. Luciferase expression was measured using luciferase assay
reagent (Promega) according to the manufacturer’s instructions.
Controls containing 1 μL of DMSO with and without HL2/3 cells
were measured for each compound, and experiments were performed in
triplicate.
was measured as previously published22 (link) and
using cell lines obtained through the NIH AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. Target cells were TZM-bl
cells (no. 8129, contributed by J.C. Kappes, X. Wu, and Tranzyme Inc.)
expressing CD4, CCR5, and CXCR4,40 (link) and
containing an integrated reporter gene for firefly luciferase under
control of HIV-1 LTR.41 (link) Effector cells
were HL2/3 (no. 1294, contributed by B. K. Felber and G. N. Pavlakis)
which produce HXB2 Env, Tat, and Rev.42 (link) Serially diluted inhibitors were added to 96-well plates containing
25 000 TZM-bl cells per well cultured overnight. 50 000
HL2/3 cells were added per well, and fusion was allowed to proceed
for 6 h in reduced serum medium (Gibco) with a final concentration
of 1% DMSO. Luciferase expression was measured using luciferase assay
reagent (Promega) according to the manufacturer’s instructions.
Controls containing 1 μL of DMSO with and without HL2/3 cells
were measured for each compound, and experiments were performed in
triplicate.
