Journal: Journal of Biological Chemistry

Article Title: Smac/DIABLO Selectively Reduces the Levels of c-IAP1 and c-IAP2 but Not That of XIAP and Livin in HeLa Cells

doi: 10.1074/jbc.m401253200

Figure Lengend Snippet: FIG. 6. Smac does not promote the auto-ubiquitination of XIAP in vitro. The purified XIAP (without tag) was incubated for 2 h at 30 °C with or without different concentrations of Smac or with Smac A in the reconstituted ubiquitination reaction system (Ub Mix) consisting of 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM Mg-ATP, 20 M mammalian ubiquitin, 100 nM rabbit E1, and 400 nM UbcH6 (E2). The reactions were stopped by adding equal volumes of 2 SDS sample loading buffer, and the products were subjected to SDS-PAGE followed by immunoblotting with anti-XIAP antibody.

Article Snippet: This paper is available on line at http://www.jbc.org 16963 at SO U T H E R N IL L IN O IS U N IV on M arch 5, 2015 http://w w w .jbc.org/ D ow nloaded from of human c-IAP2, and residues 244–263 of human XIAP, and the monoclonal antibody against human ubiquitin were purchased from R & D Systems.

Techniques: Ubiquitin Proteomics, In Vitro, Purification, Incubation, SDS Page, Western Blot

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) Lysates (300 μg) of PC3 cells (1 × 10 6 ) transfected with 2 μg mock (black) or PN1 expressing vector (white) were incubated on an array of 35 pro- and anti-apoptotic proteins. Absolute expression levels were calculated and plotted ( N = 3, one-way ANOVA, * P < 0.05). (B) XIAP and DR5 protein levels validated using immunoblotting and relative intensities measured. ( N = 3, t -test, * P < 0.05). (C) Recombinant PN1 (2 μM) or TRAIL protein (200 ng/ml) alone or in combination was added to the medium of PC3 cells (1 × 10 5 ) for 24 hrs followed by an overall cell count ( N = 3,one-way ANOVA, * P < 0.05, ** P < 0.01) (D) PC3 xenograft tumor volumes from groups pre-treated with PN1 (10 μM) or treated with daily IP of TRAIL protein (40 mg/kg), alone or in combination with PN1 pre-treatment, were measured. ( N = 5, one-way ANOVA, P < 0.05). (E) Graphical representation of treatment effects at the 12 day time point ( N = 5, one-way ANOVA, * P < 0.05).

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot, Recombinant, Cell Counting

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) PC3 cells (1 × 10 5 ) transfected with 2 μg control vector or increasing PN1 expression vector for 24 h and measurement of xiap or pn1 mRNA levels using qRT-PCR. Right : Products resolved by 1.5% agarose gel. (B) PN1 recombinant protein added to conditioned medium of PC3 cells and measurement of xiap mRNA transcripts ( N = 3, one-way ANOVA; * P < 0.05; ** P < 0.01). Below : Immunoblotting of cell conditioned medium (CM) for PN1 protein levels. (C) PC3 cells (2 × 10 5 ) were treated with 10nM negative control siRNA (Neg) or siRNA PN1 (siPN1) for 48 h and measurement xiap mRNA transcripts. Right: Immunoblotting of XIAP and PN1 protein levels in whole cell lysates (WL) and cell conditioned medium (CM).

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Recombinant, Western Blot, Negative Control

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) In PC3 (1 × 10 5 ) cells, uPA activity is reduced following transfection with a PN1 expression vector (2 μg) ( N = 4, t -test, * P < 0.01). (B) Knock-down of PN1 via (10nM) siRNA increases uPA activity ( N = 4, one-way ANOVA, * P < 0.01). Mock treatment and scrambled siRNA (NEG) used as controls. (C) 1U or 5U of recombinant uPA proteins were added to the medium of PC3 cells (1 × 10 5 ) for 24 h and xiap mRNA transcripts were measured ( N = 4, one-way ANOVA, * P < 0.05). (D) Immunoblotting of proteins from PC3 conditioned media treated with 10nM control siRNA (NEG) or siRNA uPA (siuPA) and with or without transfection of PN1 (i) and quantitation of xiap transcripts (ii). (Two-way ANOVA; N = 3, * P < 0.01). (E) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or PN1-LRP binding mutant (mLRP) or RCL binding mutant (mRCL), and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA, * P < 0.01). (F) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or treated with anti-LRP (50 μg/ml), anti-uPAR (50 μg/ml) blocking antibody for 24 h, and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA with Tukey Test, * P < 0.01; ** refer to similarly significant comparisons between specific groups as denoted by the horizontal lines over the bar graph). (G) Immunoblotting of PC3 lysates transfected with PN1 vector or treated with anti-LRP or anti-uPAR blocking antibody for 24 h.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Knockdown, Recombinant, Western Blot, Control, Quantitation Assay, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) PC3 cells (2 × 10 5 ) transfected with mock or PN1-expressing vector or treated with an NF-κB inhibitor (BAY11-7085, 5 μM), or a combination of both, and measurement of xiap mRNA ( N = 4, one-way ANOVA, * P < 0.05). (B) Blotting of NF-κB pathway members from PC3 cells following PN1 overexpression or (C) PC3 xenografts +/− pre-treatment with PN1 (10 μM) recombinant protein. (D) PC3 xenografts or xenografts pre-treated with PN1 (10 μM), DAB-stained (brown) for p65 and XIAP. Blue stain represents hemotoxylin nuclear staining.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Recombinant, Staining

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) PC3 cells (1 × 10 5 ) transfected with 2 μg of Mock or PN1 expressing vector, a PI-3k/AKT inhibitor (LY 294002, 10 μM), or a combination of both, and measurement of xiap mRNA ( N = 4, one-way ANOVA, * P < 0.05). (B) PC3 cells transfected with 2 μg Mock or PN1-expressing vectors for 24 h and blotted with indicated antibodies or (C) tissue lysates from wild type or pn1−/− mice blotted for AKT, XIAP, or XIAP-phospho-serine 87 antibodies. (D) PC3 cells transfected with 2 μg Mock or PN1 vector, treated with AKT inhibitor MK-2206 (10 ng/ml) alone, or a combination of both, and measurement of XIAP phosphorylation. (E) PC3 xenografts +/− PN1 (10 μM) were blotted via immunoblotting as well as DAB-stained (brown) for phospho-AKT (F) Blue stain represents hemotoxylin nuclear staining.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Western Blot, Staining

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) DAB-staining of PN1, p65 and XIAP in human prostatic tissue. Measurement of staining intensity comparing normal prostate tissue ( n = 12) versus a subdivision of prostate cancer into Gleason scores 6–7 ( n = 14) or 8–10 ( n = 22). One-way ANOVA performed. Correlation between groups (B–D) calculated using Spearman's r -value.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Staining

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: PN1 expression mediates apoptosis via regulation of XIAP. PN1 can affect this regulation through down-regulation of the NF-κB pathway activator p65, impeding XIAP transcription. Alternately, PN1 activity can reduce the stability of XIAP by preventing a stabilizing phosphorylation at serine-87, mediated by the AKT pathway. The dual regulation of XIAP is facilitated through inhibition of uPA and altered signalling through uPAR.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Expressing, Activity Assay, Phospho-proteomics, Inhibition

Journal: Journal of Clinical Investigation

Article Title: Targeting the XIAP/caspase-7 complex selectively kills caspase-3–deficient malignancies

doi: 10.1172/jci67951

Figure Lengend Snippet: Figure 2 I-Lys directly triggers CASP7-mediated apoptotic signaling by disrupting the XIAP:p19/p12-CASP7 complex in MCF-7 cells. (A) Determination of intracellular caspase activity in untreated (UT) MCF-7 cells or in MCF-7 cells treated with IDAM, N-acetyl-Lys, I-Lys, or STS (1 μM each) for 24 hours. (B) Immunoblotting for the full-length (FL) and cleaved forms of PARP in MCF-7 cells treated with 1 μM I-Lys for the indicated times. (C) Apoptosis in MCF-7 cells treated with 1 μM I-Lys for 24 hours in the presence or absence of the CASP7 inhibitor MPS or the PARP inhibitor IAB at the indicated doses. (D–F) Immunoblotting for pro-CASP7 and p19/p12-CASP7 (D), tracing p19/p12-CASP7 by biotin-VAD (E), and immunoprecipitation/Western blot analysis of XIAP:p19/p12-CASP7 complexes (F) in MCF-7 cells treated with I-Lys or STS (1 μM each) for the indicated times. (G) Real-time DEVDase activity in MCF-7 cells treated with I-Lys (1 μM), in the absence or presence of MPS (10 μM), or with STS (1 μM). I-Lys or STS were added to the medium 50 seconds after the onset of the experiment. Scale bar: 100 seconds. (A and C) Data are mean ± SEM.

Article Snippet: For the E. coli system, the human cDNA sequences encoding CASP3 (NM_004346.3), CASP7 (NM_033339.3), and XIAP (NM_0011167.2) from commercial cDNA clones (Origene) were used as templates to construct full-length CASP3 and CASP7 and the linker-BIR2 domain (residues 124–240) of XIAP using the sticky-end PCR method with EcoRI/ XhoI restriction sites.

Techniques: Activity Assay, Western Blot, Immunoprecipitation

Journal: Journal of Clinical Investigation

Article Title: Targeting the XIAP/caspase-7 complex selectively kills caspase-3–deficient malignancies

doi: 10.1172/jci67951

Figure Lengend Snippet: Figure 5 Anticancer effectiveness of targeting the XIAP:p19/p12-CASP7 complex in vitro and in vivo. (A) In vitro binding assay for XIAP (GST-tagged linker-BIR2 domain) interaction with the cleaved form of CASP3 or p19/p12-CASP7, in the presence of increasing I-Lys concentrations (0, 0.1, and 0.5 mM). (B) Cell apoptosis in a panel of breast cancer cell lines treated with 1 μM I-Lys at for 24 hours. Data (mean ± SEM) are from 3 indepen- dent experiments. (C) RT-PCR analysis for CASP3, CASP7, and GAPDH expression and Western blot (WB) analysis for pro-CASP3, pro-CASP7, p19/p12-CASP7, and GAPDH in various breast cancer cells. The symbol ** represents a nonspecific reaction of CASP7 antibody. (D and E) Tumor mass (D) and volume (E) from MCF-7, MDA-MB-157, and MDA-MB-231–xenografted mice treated with PBS control (n = 8) or with I-Lys at 5 (n = 6) and 25 (n = 8) mg/kg for 5 weeks. Arrows indicate the time points of I-Lys administration. *P < 0.05 versus PBS, 1-way ANOVA and Dun- can’s multiple range test. (F and G) Ki-67 immunostaining (F; brown) and TUNEL assay (G; green) of MCF-7 tumor tissues derived from mice treated without or with 25 mg/kg I-Lys. Nuclei in G were stained with DAPI (blue). Original magnification, ×200.

Article Snippet: For the E. coli system, the human cDNA sequences encoding CASP3 (NM_004346.3), CASP7 (NM_033339.3), and XIAP (NM_0011167.2) from commercial cDNA clones (Origene) were used as templates to construct full-length CASP3 and CASP7 and the linker-BIR2 domain (residues 124–240) of XIAP using the sticky-end PCR method with EcoRI/ XhoI restriction sites.

Techniques: In Vitro, In Vivo, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Control, Immunostaining, TUNEL Assay, Derivative Assay, Staining

Journal: Journal of Clinical Investigation

Article Title: Targeting the XIAP/caspase-7 complex selectively kills caspase-3–deficient malignancies

doi: 10.1172/jci67951

Figure Lengend Snippet: Figure 8 Proposed pathway for disrupting the XIAP:p19/ p12-CASP7 complex in CASP3/DR malignancies, and a chemotherapeutic strategy for targeting the XIAP:p19/p12-CASP7 complex and pro-CASP7 with I-Lys to directly kill tumor cells or sensitize CASP3/DR malignancies to chemotherapy.

Article Snippet: For the E. coli system, the human cDNA sequences encoding CASP3 (NM_004346.3), CASP7 (NM_033339.3), and XIAP (NM_0011167.2) from commercial cDNA clones (Origene) were used as templates to construct full-length CASP3 and CASP7 and the linker-BIR2 domain (residues 124–240) of XIAP using the sticky-end PCR method with EcoRI/ XhoI restriction sites.

Techniques:

Journal:

Article Title: Chemically synthesized human survivin does not inhibit caspase-3

doi: 10.1110/ps.036145.108

Figure Lengend Snippet: (A) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (Insets) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. (B) Ligated full-length survivin characterized by C18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. (C) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn2+, pH 7.5 (thick line). (D) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset. (E) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. (F) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn2+ (empty squares), and synthetic survivin in the presence of Zn2+ (filled squares). Each curve is the mean of three independent experiments.

Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R&D Systems.

Techniques: Ligation, Inhibition

Journal: Oncology reports

Article Title: Ribosomal protein S3 regulates XIAP expression independently of the NF-κB pathway in breast cancer cells.

doi: 10.3892/or.2017.6008

Figure Lengend Snippet: Figure 1. Curcumin downregulates the XIAP protein in MCF-7 breast cancer cells. (A) The MCF-7 cells were treated with the indicated concen- trations of curcumin for 72 h. The inhibition of cell growth was evaluated using the CCK-8 solution. (B) The MCF-7 cells were treated with the indicated doses of curcumin for 48 h. Apoptosis was quantified by flow cytometry. (C) Western blot analysis of the MCF-7 cells treated with cur- cumin for 24 h. Columns, means of triplicate data; bars, SD. *P<0.05. XIAP, X-linked inhibitor of apoptosis; sub-G1, hypodiploid population.

Article Snippet: The membranes were blocked in Tris-buffered saline containing 5% skim milk and incubated with primary antibodies at room temperature for 1 h. The primary antibodies were a mouse monoclonal anti-XIAP antibody (MAB822; R&D Systems, Minneapolis, MN, USA), a rabbit monoclonal anti-RPS3 antibody (#9538; Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal anti-β-actin antibody (A5441; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Inhibition, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Oncology reports

Article Title: Ribosomal protein S3 regulates XIAP expression independently of the NF-κB pathway in breast cancer cells.

doi: 10.3892/or.2017.6008

Figure Lengend Snippet: Figure 3. Knockdown of RPS3 downregulates the XIAP protein independently of the NF-κB pathway in MCF-7 breast cancer cells. (A-D) The MCF-7 cells were transfected with siRPS3 #1, siRPS3 #2, siRPS3 #3, or a negative control siRNA (siCtrl). After 120 h, cell growth (A) and apoptosis (B) were evaluated. After 96 h, the protein (C) and mRNA (D) levels of XIAP were analyzed. Columns, means of triplicate data; bars, SD. *P<0.05. XIAP, X-linked inhibitor of apoptosis; RPS3, ribosomal protein S3; sub-G1, hypodiploid population.

Article Snippet: The membranes were blocked in Tris-buffered saline containing 5% skim milk and incubated with primary antibodies at room temperature for 1 h. The primary antibodies were a mouse monoclonal anti-XIAP antibody (MAB822; R&D Systems, Minneapolis, MN, USA), a rabbit monoclonal anti-RPS3 antibody (#9538; Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal anti-β-actin antibody (A5441; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Knockdown, Transfection, Negative Control

Journal: Oncology reports

Article Title: Ribosomal protein S3 regulates XIAP expression independently of the NF-κB pathway in breast cancer cells.

doi: 10.3892/or.2017.6008

Figure Lengend Snippet: Figure 4. Knockdown of RPS3 also downregulates the XIAP protein independently of NF-κB in the MDA-MB-231 breast cancer cells. (A-D) The MDA‑MB- 231 cells were transfected with siRPS3 or control siRNA (siCtrl). After 120 h, the cell growth (A) and apoptosis (B) were assessed. After 96 h, the protein (C) and mRNA (D) levels of XIAP were assessed. Columns, means of triplicate data; bars, SD. *P<0.05. XIAP, X-linked inhibitor of apoptosis; RPS3, ribosomal protein S3; sub-G1, hypodiploid population.

Article Snippet: The membranes were blocked in Tris-buffered saline containing 5% skim milk and incubated with primary antibodies at room temperature for 1 h. The primary antibodies were a mouse monoclonal anti-XIAP antibody (MAB822; R&D Systems, Minneapolis, MN, USA), a rabbit monoclonal anti-RPS3 antibody (#9538; Cell Signaling Technology, Danvers, MA, USA) and a mouse monoclonal anti-β-actin antibody (A5441; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Knockdown, Transfection, Control

Journal: Oncotarget

Article Title: Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

doi:

Figure Lengend Snippet: (A) In PC3 (1 × 10 5 ) cells, uPA activity is reduced following transfection with a PN1 expression vector (2 μg) ( N = 4, t -test, * P < 0.01). (B) Knock-down of PN1 via (10nM) siRNA increases uPA activity ( N = 4, one-way ANOVA, * P < 0.01). Mock treatment and scrambled siRNA (NEG) used as controls. (C) 1U or 5U of recombinant uPA proteins were added to the medium of PC3 cells (1 × 10 5 ) for 24 h and xiap mRNA transcripts were measured ( N = 4, one-way ANOVA, * P < 0.05). (D) Immunoblotting of proteins from PC3 conditioned media treated with 10nM control siRNA (NEG) or siRNA uPA (siuPA) and with or without transfection of PN1 (i) and quantitation of xiap transcripts (ii). (Two-way ANOVA; N = 3, * P < 0.01). (E) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or PN1-LRP binding mutant (mLRP) or RCL binding mutant (mRCL), and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA, * P < 0.01). (F) PC3 cells transfected with control or expression vectors (2 μg) for WT-PN1 or treated with anti-LRP (50 μg/ml), anti-uPAR (50 μg/ml) blocking antibody for 24 h, and measurement of xiap expression by ELISA ( N = 4, one-way ANOVA with Tukey Test, * P < 0.01; ** refer to similarly significant comparisons between specific groups as denoted by the horizontal lines over the bar graph). (G) Immunoblotting of PC3 lysates transfected with PN1 vector or treated with anti-LRP or anti-uPAR blocking antibody for 24 h.

Article Snippet: All ELISA detection of XIAP protein was performed using the Human Total XIAP DuoSet IC (R&D Systems, DYC822) according to factory instructions.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Knockdown, Recombinant, Western Blot, Control, Quantitation Assay, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Blocking Assay