Dominant-negative ZIPK and Par-4 suppress apoptosis induced by POD-stimulating agents. 293T (5 × 10⁵) cells were transiently cotransfected with the indicated plasmids (total, 3.0 μg), together with 0.3 μg of the GFP expression plasmid. After 12 h, cells were stimulated with 1,500 U of IFN-γ/ml, 1.0 μM As₂O₃, or 100 μM STS. After 3 days (IFN-γ and As₂O₃) or 24 h (STS) of incubation, the percentages of GFP-positive apoptotic cells were determined, counting 200 cells (means ± standard deviations; n = 3).

ZIPK, Daxx, and Par-4 are necessary for apoptosis mediated by As₂O₃ and IFN-γ, as determined by using siRNA. (A) HeLa cells were transfected with siRNA targeting ZIPK, Daxx, or Par-4, and cells were analyzed by immunoblotting using anti-ZIPK, anti-Daxx, anti-Par-4, and anti-tubulin antibodies, verifying siRNA-mediated reduction in endogenous ZIPK, Daxx, and Par-4. CNTL, control. (B) HeLa cells were treated with siRNA. After 24 h, cells were stimulated with IFN-γ or As₂O₃ for 3 or 4 days. The percentages of apoptotic cells were determined by DAPI staining, counting a minimum of 300 cells (means ± standard errors; n = 3). (C) HeLa cells were treated with siRNA. After 24 h, cells were stimulated with IFN-γ or As₂O₃ for 4 days. Extracts prepared from HeLa cells or after treatment with siRNA were tested for the activation of caspase 3, based on the release of AFC from the Ac-DEVD-AFC substrate.

Par-4 facilitates the association between ZIPK and Daxx. (A) 293T cells (10⁶ cells) were transiently transfected with 2.0 μg of Myc-Par-4 together with 2.0 μg of FLAG-Daxx or FLAG-ZIPK. Cell lysates were immunoprecipitated (IP) with an anti-Myc or anti-FLAG antibody, followed by immunoblot analysis using an anti-FLAG or anti-Myc antibody, as indicated. WB, Western blotting; WCL, whole-cell lysates. (B) 293T cells (10⁶ cells) were transiently transfected with the indicated combinations of plasmids encoding Myc-Par-4, FLAG-Daxx, HA-wild-type (WT) ZIPK, HA-ZIPK K42A, or HA-ZIPK ΔLZ. Total amounts of DNA were kept at 6.0 μg. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblot analysis using an anti-FLAG antibody (top). Aliquots of whole-cell lysates were simultaneously subjected to immunoblot analysis using an anti-Myc (upper middle), anti-FLAG (lower middle), or anti-HA (bottom) antibody. (C) 293T cells (10⁶ cells) were transiently transfected with 2.5 μg of expression plasmids encoding Myc-Par-4 and FLAG-Daxx as indicated. Total amounts of DNA were held at 5.0 μg by addition of an empty vector. After 36 h, cells were lysed and immunoprecipitated with an anti-ZIPK antibody (Ab1), followed by immunoblot analysis using an anti-FLAG antibody. Aliquots of the whole-cell lysates were simultaneously subjected to immunoblot analysis using an anti-FLAG or anti-Myc antibody. (D) 293T cells (10⁶ cells) were transiently transfected with the indicated combinations of plasmids encoding FLAG-ZIPK, Myc-Par-4, and HA-Daxx, holding total amounts of DNA at 6.0 μg by addition of an empty vector. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by immunoblot analysis using an anti-FLAG antibody. Aliquots of whole-cell lysates were simultaneously subjected to immunoblot analysis using an anti-Myc, anti-FLAG, or anti-HA antibody.

Induction of cell death by ZIPK, Daxx, and Par-4. (A) 293T cells (5 × 10⁵ cells) were transiently cotransfected with the indicated combinations of plasmids (total, 3.0 μg) together with 0.3 μg of a GFP expression plasmid. After 48 h, GFP-positive cells were evaluated by microscopy, and the percentages of GFP-positive cells with apoptotic morphology were determined, counting 200 cells (means ± standard deviations [SD]; n = 3). (B) 293T cells were transiently cotransfected with the indicated combinations of plasmids (total, 3.0 μg). After 48 h, cytoplasmic proteins were extracted and caspase activity was measured, with Ac-DEVD-AFC as the substrate. Data represent relative fluorescence units (RFU) normalized for protein concentration (means ± SD; n = 3). (C) 293T cells (5 × 10⁵ cells) were transiently cotransfected with the indicated combinations of plasmids (total, 3.0 μg) together with 0.3 μg of GFP expression plasmids. After 48 h, the percentages of GFP-positive apoptotic cells were determined, counting 200 cells (means ± SD; n = 3).

ZIPK K42A prevents Daxx translocation into PODs in response to IFN-γ and As₂O₃. (A) HeLa cells were transiently transfected with FLAG-ZIPK K42A. After 36 h of transfection, cells were stimulated with As₂O₃ or IFN-γ for 6 or 18 h, respectively. The cells were fixed and stained sequentially with an anti-FLAG antibody (top), an anti-Daxx antibody (middle), and DAPI (bottom). Arrows, cells expressing FLAG-ZIPK K42A. Note that Daxx translocation to PODs was prevented by expression of ZIPK K42A. (B) Cells were prepared as for panel A. The percentage of transfected cells with Daxx translocation to PODs is presented, counting at least 200 transfected cells from three independent experiments (means ± standard deviations [SD]). CNTL, control; WT, wild type. (C) HeLa cells were transiently transfected with FLAG-ZIPK K42A. After 36 h of transfection, cells were stimulated with As₂O₃ or IFN-γ for 6 or 18 h, respectively. The cells were fixed and stained sequentially with an anti-FLAG antibody (top), an anti-PML antibody (middle), and DAPI (bottom). Arrows, cells expressing FLAG-ZIPK K42A. Note that PML translocation to PODs was not affected by expression of ZIPK K42A. (D) Cells were prepared as for panel C. Shown are the percentages of cells with PML translocation to PODs, counting at least 200 transfected cells from three independent experiments (means ± SD).

ZIPK associates with Daxx. (A) One million 293T cells were transiently transfected with 2.0 μg of plasmid DNA encoding FLAG-tagged Daxx. After 36 h, cell lysates were immunoprecipitated (IP) with control rabbit anti-mouse Ig serum, anti-ZIPK (Ab1), or another anti-ZIPK antibody (Ab2). Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with an anti-FLAG monoclonal antibody (top). Aliquots of whole-cell lysates (WCL) were simultaneously subjected to immunoblot analysis using an anti-FLAG antibody (bottom). WB, Western blotting. (B) 293T cells were transiently transfected with the indicated combinations of HA-Daxx (FL), HA-Daxx ΔC (ΔC), FLAG-ZIPK, and FLAG-Tpl2. Total amounts of plasmids were kept constant at 6.0 μg by supplementation with the empty pcDNA3 plasmid. After 36 h, cell lysates were immunoprecipitated with an anti-HA monoclonal antibody, followed by Western blotting with an anti-FLAG antibody (top). Aliquots of whole-cell lysates were simultaneously subjected to immunoblot analysis using an anti-FLAG (middle) or anti-HA antibody (bottom). (C) HeLa cells (3 × 10⁸) were stimulated with IFN-γ or As₂O₃ for 12 h. Cell lysates were prepared, and immunoprecipitated with a monoclonal Daxx or control (CNTL) antibody. The immunoprecipitates were separated by SDS-PAGE, followed by blotting with an anti-ZIPK antibody (Ab1). Aliquots of the whole-cell lysates were simultaneously subjected to Western blot analysis using an anti-Daxx or anti-ZIPK antibody as indicated. N, nonstimulated; I, IFN-γ; A, As₂O₃.

ZIPK colocalizes with Daxx in PODs. (A) HeLa cells grown on coverslips were transiently transfected with FLAG-ZIPK. After 36 h of transfection, cells were stimulated with 1,500 U of IFN-γ/ml or 1.0 μM As₂O₃ for 12 h. After fixation, cells were stained with an anti-FLAG antibody (M2) for the detection of ZIPK and DAPI for visualization of nuclei. Antibody detection was achieved with a FITC-conjugated anti-mouse antibody (green), followed by microscopy analysis. The bottom panels show the overlay result. (B) HeLa cells transiently transfected with FLAG-ZIPK were stimulated with IFN-γ and As₂O₃ for 12 h and stained with an anti-FLAG antibody, and the percentage of transfected cells with a speckled pattern was determined by counting a minimum of 200 transfected cells. Data represent means ± standard deviations (n = 3). (C and D) HeLa cells transiently transfected with FLAG-ZIPK were stimulated with IFN-γ or As₂O₃ for 12 h and stained sequentially with an anti-FLAG monoclonal antibody (top), an anti-PML polyclonal antibody (C, upper middle), or an anti-Daxx polyclonal antibody (D, upper middle) and DAPI (bottom). Antibody detection was achieved with a FITC-conjugated anti-mouse antibody (green) and a rhodamine-conjugated anti-rabbit antibody (red). Overlay results (merge) demonstrate colocalization of FLAG-ZIPK and PML or Daxx in response to IFN-γ and As₂O₃.

As₂O₃ and IFN-γ induce POD formation and apoptosis. (A) Immunoblot analysis of ZIPK, Par-4, and Daxx in HeLa cells was performed using cell lysates normalized for total protein content (50 μg per lane) and specific antibodies that detect the endogenous proteins. Cells were stimulated for the indicated times with 1,500 U of IFN-γ/ml or 1.0 μM As₂O₃ prior to lysis. (B) HeLa cells stimulated for 12 h with IFN-γ or As₂O₃ were fixed and stained with an anti-PML monoclonal antibody and a FITC-conjugated anti-mouse IgG secondary antibody (top) and then stained with an anti-Daxx polyclonal antibody and a rhodamine-conjugated anti-rabbit secondary antibody (upper middle) and DAPI (bottom). Overlay results (merge) demonstrate colocalization of PML and Daxx in response to IFN-γ and As₂O₃. Cells were imaged by fluorescence microscopy. (C) HeLa cells were treated with IFN-γ and As₂O₃ for the indicated periods. The percentage (Å) of viable cells was determined by DAPI staining, counting a minimum of 300 cells (means ± standard errors; n = 3). (D) HeLa cells were stimulated with As₂O₃ or IFN-γ for 48 or 72 h. Cytoplasmic proteins were extracted, and the caspase activity was measured, with Ac-DEVD-AFC as the substrate. RFU, relative fluorescence units.

ZIPK binds and phosphorylates Par-4. (A) Cell lysates prepared from HeLa cells (3 × 10⁸) were divided into equal volumes and used for immunoprecipitation (IP) with an anti-Par-4 monoclonal antibody (P) or control IgG (C), followed by immunoblotting with an anti-ZIPK antibody (Ab1). Aliquots of whole-cell lysates (W) were simultaneously subjected to immunoblot analysis using an anti-ZIPK antibody. WB, Western blotting. Arrowhead, band corresponding to ZIPK. (B) Plasmids expressing ZIPK or mutant versions of it fused to the GAL4 DNA-binding domain were cotransformed with a plasmid expressing Par-4 or various mutant proteins fused to the GAL4 trans-activation domain. Interactions were detected on the basis of the ability of cells to grow on medium lacking leucine, tryptophan, and histidine (+). β-Gal, β-galactosidase. (C) COS-7 cells were transiently transfected with the indicated combinations of plasmids encoding FLAG-ZIPK, Myc-wild-type Par-4 (WT), Myc-Par-4 ΔLZ (ΔLZ), or Myc-Par-4 LZ (LZ), holding the amounts of DNA at 5.0 μg by addition of an empty vector. Cell lysates were immunoprecipitated with an anti-Myc antibody (9E10), followed by immunoblot analysis using an anti-FLAG antibody (M2) as indicated. (D) COS-7 cells were transiently transfected with the indicated combinations of plasmids. After 36 h, cell lysates were immunoprecipitated with an anti-Myc antibody and subjected to in vitro kinase assays. Aliquots of whole-cell lysates were simultaneously subjected to immunoblot analysis using an anti-FLAG or anti-Myc antibody. IVK, in vitro kinase assay. (E) HeLa cells grown on coverslips were transiently transfected with Myc-Par-4. At 36 h after transfection, cells were stained with an anti-Myc monoclonal antibody. Antibody detection was achieved by using a FITC-conjugated anti-mouse antibody.