Fig. 1.
Fig. 1. Caspase 3 and PARP were cleaved in response to IFN-γ and anti-fas antibody treatment, but caspase 1 was not. / Immunoblots and fluorogenic assays on lysates from JY (EBV-transformed normal B cells) and HSC536N (EBV-transformed FA-C) cells exposed to 100 ng/mL agonistic anti-fas antibody and 1.0 ng/mL IFN-γ for various times describe the sequential activation of caspase 3 and PARP in these cells. (A) Samples were separated by 12% SDS-PAGE and immunoblotted using a monoclonal caspase 3 antibody. The antibody detects procaspase 3 as well as the p17 cleaved form of caspase 3. The cleaved forms of caspase 3 were detectable by 60 minutes in both normal and FA cells. The 2-hour samples were run on a separate gel due to space constraints. (B) Immunoblot from a 7.5% SDS-PAGE probed with a monoclonal PARP antibody. The antibody detects p116 PARP and the p85 cleavage product. P85 PARP was present in both FA and normal cells after 3 hours of treatment with 100 ng/mL agonistic anti-fas antibody and 1.0 ng/mL IFN-γ. (C) Immunoblot of JY and HSC536N lymphoblast cell lysates probed with a caspase 1 antibody. The antibody detects the cleaved (p10) form of caspase 1. Total cellular protein (100 μg) was loaded per lane, and proteins were separated on 15% SDS-PAGE. Ten- and 120-minute samples were also immunoblotted, and showed no induction over constitutive levels (data not shown). (D) Fluorogenic assays revealed no activation of caspase 1, but early (60 minutes) activation of caspase 3, which reached maximal activation by 120 minutes. The maximal level of 275 fluorescence units (FU) caspase 3 represents 240 ng/μg total protein, as determined from a standard curve generated using human recombinant caspase 3. (E) Flow cytometry reveals constitutive caspase 3 activation, which increases with IFN-γ and anti-fas antibody treatment. There is substantially more activation of caspase 3 in the mutant lymphoblasts than in the FANCC-corrected cells both with and without this treatment. Panels i through iv represent actual flow cytometric data, with fluorescent staining of active caspase 3 shown on the y-axis (FL2-H). In all cases, 10 000 events are plotted. (i) Unexposed HSC536N cells. (ii) Unexposed HSC536N FANCC/neo cells. (iii) HSC536N cells exposed to 1 ng/mL IFN-γ and 100 ng/mLl activating anti-fas antibody for 3 hours. (iv) HSC536N FANCC/neo cells exposed to IFN-γ and anti-fas antibody under the same conditions. (v) Combined data from 4 separate experiments with the percentage of cells positively staining for active caspase 3 on the y-axis. The arbitrary cutoff line for positive staining is represented in panels i through iv by the horizontal line across each plot and is placed identically in each. Bar 1 shows unexposed HSC536N cells. Bar 2 shows HSC536N cells exposed to 1 ng/mL IFN-γ and 100 ng/mL activating anti-fasantibody for 3 hours. Bar 3 shows unexposed HSC536N FANCC/neo cells. Bar 4 shows HSC536N FANCC/neo cells exposed to IFN-γ and anti-fas antibody.

Caspase 3 and PARP were cleaved in response to IFN-γ and anti-fas antibody treatment, but caspase 1 was not.

Immunoblots and fluorogenic assays on lysates from JY (EBV-transformed normal B cells) and HSC536N (EBV-transformed FA-C) cells exposed to 100 ng/mL agonistic anti-fas antibody and 1.0 ng/mL IFN-γ for various times describe the sequential activation of caspase 3 and PARP in these cells. (A) Samples were separated by 12% SDS-PAGE and immunoblotted using a monoclonal caspase 3 antibody. The antibody detects procaspase 3 as well as the p17 cleaved form of caspase 3. The cleaved forms of caspase 3 were detectable by 60 minutes in both normal and FA cells. The 2-hour samples were run on a separate gel due to space constraints. (B) Immunoblot from a 7.5% SDS-PAGE probed with a monoclonal PARP antibody. The antibody detects p116 PARP and the p85 cleavage product. P85 PARP was present in both FA and normal cells after 3 hours of treatment with 100 ng/mL agonistic anti-fas antibody and 1.0 ng/mL IFN-γ. (C) Immunoblot of JY and HSC536N lymphoblast cell lysates probed with a caspase 1 antibody. The antibody detects the cleaved (p10) form of caspase 1. Total cellular protein (100 μg) was loaded per lane, and proteins were separated on 15% SDS-PAGE. Ten- and 120-minute samples were also immunoblotted, and showed no induction over constitutive levels (data not shown). (D) Fluorogenic assays revealed no activation of caspase 1, but early (60 minutes) activation of caspase 3, which reached maximal activation by 120 minutes. The maximal level of 275 fluorescence units (FU) caspase 3 represents 240 ng/μg total protein, as determined from a standard curve generated using human recombinant caspase 3. (E) Flow cytometry reveals constitutive caspase 3 activation, which increases with IFN-γ and anti-fas antibody treatment. There is substantially more activation of caspase 3 in the mutant lymphoblasts than in the FANCC-corrected cells both with and without this treatment. Panels i through iv represent actual flow cytometric data, with fluorescent staining of active caspase 3 shown on the y-axis (FL2-H). In all cases, 10 000 events are plotted. (i) Unexposed HSC536N cells. (ii) Unexposed HSC536N FANCC/neo cells. (iii) HSC536N cells exposed to 1 ng/mL IFN-γ and 100 ng/mLl activating anti-fas antibody for 3 hours. (iv) HSC536N FANCC/neo cells exposed to IFN-γ and anti-fas antibody under the same conditions. (v) Combined data from 4 separate experiments with the percentage of cells positively staining for active caspase 3 on the y-axis. The arbitrary cutoff line for positive staining is represented in panels i through iv by the horizontal line across each plot and is placed identically in each. Bar 1 shows unexposed HSC536N cells. Bar 2 shows HSC536N cells exposed to 1 ng/mL IFN-γ and 100 ng/mL activating anti-fasantibody for 3 hours. Bar 3 shows unexposed HSC536N FANCC/neo cells. Bar 4 shows HSC536N FANCC/neo cells exposed to IFN-γ and anti-fas antibody.

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