Figure 6. S. aureus Hemolysin induces necrotic cell death in THP-1 cells.

Lysates from THP-1 cells that were untreated (N.T.) or treated with either Hla (1.0 µg/ml) or staurosporine (1 µM) for 4 hours were subjected to SDS-PAGE and immunoblot with antibodies directed to active caspase-3, Poly ADP ribose polymerase (PARP), or actin. Representative data from 2 independent experiments are shown. (B) THP-1 cells were untreated (N.T.) or treated with either Hla (1.0 µg/ml) or staurosporine (1 µM) over a 17 hour time course. At indicated times the cells were fixed and stained for DNA content with propidium iodide and analyzed using flow cytometry. Cell counts are plotted against propidium iodide fluorescence, which indicates DNA content of the cell. The bars on the histograms indicate the gate for subG1 DNA content (apoptotic cells). Histograms for cells treated for 4 hours are shown, the percent of cells containing subG1 DNA content at 4, 8, 17 hours are charted below the histograms. (C) THP-1 cells, untreated (left) or treated with either Hla (1.0 µg/ml, middle) or staurosporine (1 µM, right), were processed and examined by transmission electron microscopy. Normal cellular morphology is demonstrated in the untreated cells. Morphologic features of necrotic cell death including loss of plasma membrane integrity and cytoplasmic contents as well as intact nuclei with loose chromatin are demonstrated in the Hla treated cells. Apoptotic morphology with nuclear condensation and apoptotic body formation is demonstrated in a staurosporine-treated cell. (D) Culture supernatants from THP-1 cells that were untreated or treated with either Hla (1.0 µg/ml) or staurosporine (1 µM)for 1 and 6 hours were subjected to SDS-PAGE and immunoblot against HMGB1 (left panel). Cell pellets were analyzed for PARP cleavage using immunoblot (right panel). Representative data from at least 3 independent experiments are shown.