Figure 5
Figure 5. PLT membrane protein induces apoptosis via membrane-bound FasL. (A) MEFs were incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of membrane proteins (membrane fraction) of buffer- or ADP-stimulated PLTs from WT mice or FasL△m/△m mice (lacking membrane-bound FasL only) for 6 hours. Isolated soluble proteins (soluble fraction) of buffer- or ADP-stimulated PLTs from WT mice or FasL△m/△m mice were incubated in a dilution of 5 × 10−4 of the protein sample. Subsequently, LDH activity was measured. Data represent mean ± SEM (n = 9) and are shown as percentage of total cellular LDH levels of untreated cells. *P < .05 vs FasL△m/△m, soluble- and membrane-fraction of resting WT PLTs. (B) Caspase 3/7 activity kinetics were measured in MEFs incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of the isolated membrane protein fraction of ADP-stimulated PLTs from WT or FasL△m/△m mice for 6 hours. Isolated soluble proteins (soluble fraction) were incubated in a dilution to 5 × 10−4 of the protein sample. Data were normalized to the corresponding samples from resting PLTs. Data are shown as percentage of control and represent mean ± SEM (n = 6). *P < .05 vs FasL△m/△m or soluble fractions. (C) LDH activity was measured after 6 hours in the supernatant of mitochondrial apoptosis-incompetent Bax/Bak DKO MEFs incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of membrane protein (membrane fraction) of resting or ADP-stimulated PLTs from WT or FasL△m/△m mice. Soluble proteins (soluble fraction) were incubated in a dilution to 5 × 10−4 of the isolated proteins. Data represent mean ± SEM (n = 6) and are shown as percentage of total cellular LDH levels of untreated cells. *P < .05 vs FasL△m/△m, soluble fraction or membrane fraction of resting WT PLTs. (D) SH-SY5Y cells transfected with GFP-Bax were incubated with resting or ADP-stimulated and PFA-fixed PLTs for 6 hours. Co-localization of GFP-Bax fluorescence (green) with mitochondrial Tom20 staining (red) is shown in yellow in the merged panel. Bax activation was detected with the conformation-specific anti-Bax Ab 6A7. Co-localization of active Bax and mitochondrial Bax is shown in cyan and white, respectively, in the merged panel. Bars, 10 µm.

PLT membrane protein induces apoptosis via membrane-bound FasL. (A) MEFs were incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of membrane proteins (membrane fraction) of buffer- or ADP-stimulated PLTs from WT mice or FasL△m/△m mice (lacking membrane-bound FasL only) for 6 hours. Isolated soluble proteins (soluble fraction) of buffer- or ADP-stimulated PLTs from WT mice or FasL△m/△m mice were incubated in a dilution of 5 × 10−4 of the protein sample. Subsequently, LDH activity was measured. Data represent mean ± SEM (n = 9) and are shown as percentage of total cellular LDH levels of untreated cells. *P < .05 vs FasL△m/△m, soluble- and membrane-fraction of resting WT PLTs. (B) Caspase 3/7 activity kinetics were measured in MEFs incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of the isolated membrane protein fraction of ADP-stimulated PLTs from WT or FasL△m/△m mice for 6 hours. Isolated soluble proteins (soluble fraction) were incubated in a dilution to 5 × 10−4 of the protein sample. Data were normalized to the corresponding samples from resting PLTs. Data are shown as percentage of control and represent mean ± SEM (n = 6). *P < .05 vs FasL△m/△m or soluble fractions. (C) LDH activity was measured after 6 hours in the supernatant of mitochondrial apoptosis-incompetent Bax/Bak DKO MEFs incubated with various dilutions (4 × 10−4; 4.5 × 10−4; 5 × 10−4) of membrane protein (membrane fraction) of resting or ADP-stimulated PLTs from WT or FasL△m/△m mice. Soluble proteins (soluble fraction) were incubated in a dilution to 5 × 10−4 of the isolated proteins. Data represent mean ± SEM (n = 6) and are shown as percentage of total cellular LDH levels of untreated cells. *P < .05 vs FasL△m/△m, soluble fraction or membrane fraction of resting WT PLTs. (D) SH-SY5Y cells transfected with GFP-Bax were incubated with resting or ADP-stimulated and PFA-fixed PLTs for 6 hours. Co-localization of GFP-Bax fluorescence (green) with mitochondrial Tom20 staining (red) is shown in yellow in the merged panel. Bax activation was detected with the conformation-specific anti-Bax Ab 6A7. Co-localization of active Bax and mitochondrial Bax is shown in cyan and white, respectively, in the merged panel. Bars, 10 µm.

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