Figure 4
Figure 4. Modulation and release of FKN during apoptosis of BL cells. (A) Loss of FKN labeling from the surface of BL cells after induction of apoptosis. BL2 or Mutu-BL cells were labeled by indirect immunofluorescence using anti-FKN mAb (clone 81513) at the indicated times after induction of apoptosis by UV irradiation. Flow cytometric histograms of surface FKN on “viable-zone” cells (“Fluorescence labeling and flow cytometry”). Black represents time 0; white, 2 hours; coarse stippling, 4 hours; fine stippling, 6 hours after irradiation; light gray, background immunostaining. In the experiments shown, levels of apoptosis (assessed using annexin V) were as follows: 2%, 7%, 14%, and 41% for Mutu-BL cells at 0, 2, 4, and 6 hours, respectively; 4%, 24%, 34%, and 56% for BL2 cells at 0, 2, 4, and 6 hours, respectively. Representative experiments from at least 3 in each case. (B) Flow cytometric dot plots of viable-zone BL2 cells showing FKN (PE) (monoclonal antibody, clone 51637) vs annexin V (FITC) labeling at 0, 2, and 4 hours after UV induction. Values indicate percentage total events in each quadrant. Note that acquisition of annexin V positivity observed after UV induction accompanies loss of surface FKN labeling. Experiment shown is representative of at least 3. (C) Immunoblots of whole cell lysates showing changes in FKN after induction of apoptosis in BL2 and BL2/Bcl-2 transfectant cell lines. Blots were performed exactly as described in Figure 2C on samples prepared at the indicated times (0-3 hours) after culture in the presence or absence of staurosporine (1 μM). Full-length recombinant human FKN (rFKN) that excluded the transmembrane and cytoplasmic domains of FKN was included for comparison (50 ng/lane). Cells were monitored for induction of apoptosis and membrane integrity using annexin V and PI. In the experiment shown, cells were more than 90% apoptotic by 2 hours. (D) Loss of FKN labeling from the surface of BL cells after induction of apoptosis. L3055 and BL2 cells were labeled by indirect immunofluorescence using anti-FKN mAb as in panel A at the indicated times after induction of apoptosis by treatment with UV irradiation, actinomycin D (1 μg/mL), or staurosporine (1 μM). Flow cytometric histograms of surface FKN staining on viable-zone cells (“Fluorescence labeling and flow cytometry”) are shown. Black represents time 0; white, 1 hour; coarse stippling, 2 hours; fine stippling, 4 hours after stimulation; light gray, background immunostaining. Levels of apoptosis (assessed using annexin V) after 4 hours were: 46%, 51%, and 64% for L3055 cells treated with UV irradiation, actinomycin D, and staurosporine, respectively; 59% for actinomycin D-treated BL2. Representative experiments from at least 3 in each case. (E) Apoptosis-induced loss of FKN labeling from the surface of BL cells and blockade by the pan-caspase inhibitor zVAD-fmk and by Bcl-2. BL2 cells and Bcl-2 transfectants, BL2/Bcl-2, were labeled using anti-FKN mAb as in panel A at 2 hours after induction of apoptosis by treatment with staurosporine (1 μM) ± z-VAD-fmk (100 μM). Flow cytometric histograms of surface FKN on viable-zone cells (“Fluorescence labeling and flow cytometry”) are shown. Black represents BL cells alone; coarse stippling, BL cells treated with staurosporine; fine stippling, BL2 cells treated with staurosporine and z-VAD-fmk; pale gray, background immunostaining. Levels of apoptosis (assessed using annexin V) achieved by 2 hours in the experiments shown were: 10%, 54%, and 13% for BL2 cells alone, BL2 cells treated with staurosporine, and BL2 cells treated with staurosporine plus z-VAD-fmk, respectively; 3% and 8% for BL2/Bcl-2 cells alone and BL2/Bcl-2 cells treated with staurosporine, respectively. Representative experiments from at least 3 in each case. (F) Appearance of FKN in cell-free supernatants of BL cells induced to undergo apoptosis: (i) equal numbers of BL2 and BL2/Bcl-2 cells that had been grown in serum-free RPMI 1640 medium were induced into apoptosis for 3 hours with staurosporine treatment or after UV induction, and FKN was detected by immunoblotting of TCA/acetone-precipitated cell-free supernatants using anti-FKN mAb (clone 81513); and (ii) cell-free supernatant was collected from equal numbers of staurosporine-treated BL2 cells that were cultured in the presence or absence of z-VAD-fmk at the times indicated (1-3 hours) after treatment. FKN was detected by immunoblotting of TCA/acetone-precipitated cell-free supernatants using anti-FKN mAb (clone 81513). (G) Appearance of FKN in microparticles from BL2 cells induced to undergo apoptosis. BL2 cells were induced to undergo apoptosis using staurosporine (1 μM) and whole cell lysates (cell lysate), cell-free supernatants (cell supe), microparticle lysates (MP lysate), collected by ultracentrifugation of cell-free supernatants, and microparticle supernatants (MP supe) were prepared at up to 4 hours after treatment as indicated. Cells were monitored for induction of apoptosis and membrane integrity using annexin V and PI. In the experiment shown, cells were 90% annexin V–positive, PI-negative by 2 hours; PI positivity remained unchanged until 4 hours after treatment. FKN was detected by immunoblotting of cell lysate (30 μg of protein per lane, as indicated using a Bradford protein assay), TCA/acetone-precipitated cell-free supernatants (equal volumes of sample loaded per well), microparticle lysate (equal volumes of sample loaded per well), or TCA/acetone-precipitated microparticle supernatant (equal volumes of sample loaded per well) using anti-FKN mAb (clone 81513). Blots were stripped and reprobed for β-actin to check for loading variability.

Modulation and release of FKN during apoptosis of BL cells. (A) Loss of FKN labeling from the surface of BL cells after induction of apoptosis. BL2 or Mutu-BL cells were labeled by indirect immunofluorescence using anti-FKN mAb (clone 81513) at the indicated times after induction of apoptosis by UV irradiation. Flow cytometric histograms of surface FKN on “viable-zone” cells (“Fluorescence labeling and flow cytometry”). Black represents time 0; white, 2 hours; coarse stippling, 4 hours; fine stippling, 6 hours after irradiation; light gray, background immunostaining. In the experiments shown, levels of apoptosis (assessed using annexin V) were as follows: 2%, 7%, 14%, and 41% for Mutu-BL cells at 0, 2, 4, and 6 hours, respectively; 4%, 24%, 34%, and 56% for BL2 cells at 0, 2, 4, and 6 hours, respectively. Representative experiments from at least 3 in each case. (B) Flow cytometric dot plots of viable-zone BL2 cells showing FKN (PE) (monoclonal antibody, clone 51637) vs annexin V (FITC) labeling at 0, 2, and 4 hours after UV induction. Values indicate percentage total events in each quadrant. Note that acquisition of annexin V positivity observed after UV induction accompanies loss of surface FKN labeling. Experiment shown is representative of at least 3. (C) Immunoblots of whole cell lysates showing changes in FKN after induction of apoptosis in BL2 and BL2/Bcl-2 transfectant cell lines. Blots were performed exactly as described in Figure 2C on samples prepared at the indicated times (0-3 hours) after culture in the presence or absence of staurosporine (1 μM). Full-length recombinant human FKN (rFKN) that excluded the transmembrane and cytoplasmic domains of FKN was included for comparison (50 ng/lane). Cells were monitored for induction of apoptosis and membrane integrity using annexin V and PI. In the experiment shown, cells were more than 90% apoptotic by 2 hours. (D) Loss of FKN labeling from the surface of BL cells after induction of apoptosis. L3055 and BL2 cells were labeled by indirect immunofluorescence using anti-FKN mAb as in panel A at the indicated times after induction of apoptosis by treatment with UV irradiation, actinomycin D (1 μg/mL), or staurosporine (1 μM). Flow cytometric histograms of surface FKN staining on viable-zone cells (“Fluorescence labeling and flow cytometry”) are shown. Black represents time 0; white, 1 hour; coarse stippling, 2 hours; fine stippling, 4 hours after stimulation; light gray, background immunostaining. Levels of apoptosis (assessed using annexin V) after 4 hours were: 46%, 51%, and 64% for L3055 cells treated with UV irradiation, actinomycin D, and staurosporine, respectively; 59% for actinomycin D-treated BL2. Representative experiments from at least 3 in each case. (E) Apoptosis-induced loss of FKN labeling from the surface of BL cells and blockade by the pan-caspase inhibitor zVAD-fmk and by Bcl-2. BL2 cells and Bcl-2 transfectants, BL2/Bcl-2, were labeled using anti-FKN mAb as in panel A at 2 hours after induction of apoptosis by treatment with staurosporine (1 μM) ± z-VAD-fmk (100 μM). Flow cytometric histograms of surface FKN on viable-zone cells (“Fluorescence labeling and flow cytometry”) are shown. Black represents BL cells alone; coarse stippling, BL cells treated with staurosporine; fine stippling, BL2 cells treated with staurosporine and z-VAD-fmk; pale gray, background immunostaining. Levels of apoptosis (assessed using annexin V) achieved by 2 hours in the experiments shown were: 10%, 54%, and 13% for BL2 cells alone, BL2 cells treated with staurosporine, and BL2 cells treated with staurosporine plus z-VAD-fmk, respectively; 3% and 8% for BL2/Bcl-2 cells alone and BL2/Bcl-2 cells treated with staurosporine, respectively. Representative experiments from at least 3 in each case. (F) Appearance of FKN in cell-free supernatants of BL cells induced to undergo apoptosis: (i) equal numbers of BL2 and BL2/Bcl-2 cells that had been grown in serum-free RPMI 1640 medium were induced into apoptosis for 3 hours with staurosporine treatment or after UV induction, and FKN was detected by immunoblotting of TCA/acetone-precipitated cell-free supernatants using anti-FKN mAb (clone 81513); and (ii) cell-free supernatant was collected from equal numbers of staurosporine-treated BL2 cells that were cultured in the presence or absence of z-VAD-fmk at the times indicated (1-3 hours) after treatment. FKN was detected by immunoblotting of TCA/acetone-precipitated cell-free supernatants using anti-FKN mAb (clone 81513). (G) Appearance of FKN in microparticles from BL2 cells induced to undergo apoptosis. BL2 cells were induced to undergo apoptosis using staurosporine (1 μM) and whole cell lysates (cell lysate), cell-free supernatants (cell supe), microparticle lysates (MP lysate), collected by ultracentrifugation of cell-free supernatants, and microparticle supernatants (MP supe) were prepared at up to 4 hours after treatment as indicated. Cells were monitored for induction of apoptosis and membrane integrity using annexin V and PI. In the experiment shown, cells were 90% annexin V–positive, PI-negative by 2 hours; PI positivity remained unchanged until 4 hours after treatment. FKN was detected by immunoblotting of cell lysate (30 μg of protein per lane, as indicated using a Bradford protein assay), TCA/acetone-precipitated cell-free supernatants (equal volumes of sample loaded per well), microparticle lysate (equal volumes of sample loaded per well), or TCA/acetone-precipitated microparticle supernatant (equal volumes of sample loaded per well) using anti-FKN mAb (clone 81513). Blots were stripped and reprobed for β-actin to check for loading variability.

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