Anti-Fas- and etoposide-induced apoptosis proceed through a common downstream pathway. (A) Wild-type Jurkat cells were incubated with 100 ng/mL 7C11 anti-Fas ( — ) or 68 μmol/L etoposide (- - - -) for the indicated times at 37°C. After DNA was stained with propidium iodide, hypodiploid cells were detected by flow cytometry. In additional experiments, appearance of Jurkat cells with subdiploid DNA content was observed after 6 hours of etoposide treatment at concentrations as low as 6.8 μmol/L (Fig 4B). (B) RT-PCR analysis of caspase expression in untreated Jurkat cells. Each lane contains one-tenth volume from the PCR reaction for the target mRNA indicated above the respective lane. Except caspases-1 and -5, which were amplified for 35 cycles, all reactions were amplified for 30 cycles. The lane marked β-actin (-RT) is a control PCR reaction using poly A⁺ RNA as template. The lack of a product in this lane confirms that the products observed in the other lanes are not derived from genomic DNA. (C through E) Cells were incubated with 100 ng/mL 7C11 anti-Fas or 68 μmol/L etoposide for the indicated times at 37°C. (C) Total cellular protein was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies that recognize PARP, laminB₁ , and procaspase-2, -3, and -7. Arrowheads indicate specific cleavage products.

(D) Cytosolic extracts were assayed for DEVD-AFC, VEID-AFC, and YVAD-AFC cleavage activities. Control experiments indicated that YVAD-AFC cleavage activity could be readily detected in cytosol from THP.1 cells. (E) Cytosolic extracts were incubated with 1 μmol/L Z-EK (bio)D-amok, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with peroxidase-conjugated streptavidin. Lane 1, cytosol from etoposide-treated HL-60 cells showing the previously described IRPs that label with Z-EK(bio)D-amok.¹¹