p38 MAPK activation is required for HIV-induced FasL expression. (A) HIV-1-mediated FasL induction in HIV-1-infected cells. Quantification of FasL expression in human PBMCs infected with HIV-1 NL4-3 virus. Cell lysates or supernatants were prepared at the indicated times after infections. As a control, mock cells were stimulated with phorbol myristate acetate (PMA, 10 ng/mL) plus ionomycin (500 ng/mL) to induce FasL expression as described in “Materials and methods.” Results are expressed in concentration to demonstrate that the assay detects both forms of FasL. Data represent the mean ± standard deviation from triplicate samples derived from 1 of 3 independent experiments. (B) Immunoblot analysis of FasL expression. Total cellular proteins were prepared from the groups described in panel A. Protein lysates were blotted with anti-FasL-specific antibody or anti-p24^gag (bottom) to confirm the FasL expression and monitor viral infection status. (C-D) FasL induction is blocked by p38 inhibitors. PBMCs (C) or Jurkat T cells (D) were infected with HIV-1 NL4-3 virus in the presence or absence of 1 μM p38 inhibitor RWJ67657. At 48 hours after infection, FasL expression was measured by flow cytometry, and analysis was performed on a gated low forward scatter and CD24(HSA)-positive cells using FasL-specific antibody. It is noteworthy that the p38 inhibitor strongly blocks FasL induction in both cell types tested. Data shown are representative of 1 of 3 independent experiments. Histograms show the indicated surface marker, and filled histograms represent the isotype-matched control antibodies.