Time-course of ALP-induced apoptosis and coclustering of membrane rafts and Fas/CD95 in edelfosine- and perifosine-treated MM cells. (A) MM144 cells were incubated with 10 μM edelfosine or perifosine for the indicated times, and the proportion of cells in the sub-G1 region, representing apoptotic cells, was quantitated by flow cytometry. Data shown are means ± SD of 3 independent determinations. (B) MM144 cells were treated with 10 μM edelfosine for the indicated times and analyzed by immunoblotting with an anti–caspase-3 antibody that recognized only the p18 subunit of active caspase-3 and with an anti-PARP antibody that detected both the 116-kDa intact form of PARP and its p85 cleaved form. Data shown are representative of 3 experiments performed. (C) MM144 cells were either untreated (control) or treated with 10 μM edelfosine or perifosine for 12 hours and then stained with FITC-CTx B subunit to identify lipid rafts (green fluorescence) and with a specific anti-Fas/CD95 monoclonal antibody, followed by CY3-conjugated anti–mouse Ig antibody (red fluorescence). Areas of colocalization between membrane rafts and Fas/CD95 in the merge panels are yellow. Images shown are representative of 3 independent experiments. Bar, 10 μm.
