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: Journal of Biological Chemistry

Article Title: Down-regulation of Survivin in Nitric Oxide-induced Cell Growth Inhibition and Apoptosis of the Human Lung Carcinoma Cells

doi: 10.1074/jbc.m312381200

Figure Lengend Snippet: FIG. 2. Effects of NO donors on the levels of survivin protein and gene expression. A, the total protein extracts in a variety of cell lines were prepared for Western blot analysis using anti-survivin and ani-ERK-2 antibodies. B, H1299 cells were treated with 0.4 mM SNAP for 24 h. The cells were incubated with rabbit anti-survivin antibody and then incubated with goat anti-rabbit Cy5. The survivin protein displayed red fluorescence with goat anti-rabbit Cy5. The nuclei were stained with Hoechst 33258, which displayed blue fluorescence. C, CL3 and H1299 cells were treated with 0–2 mM SNP for 24 h, and whole cell extracts were prepared and subjected to immunoblot analysis. The relative protein levels under each treatment were the average of 3–9 independent experiments. D, CL3 cells were treated with 0.4–0.8 mM SNAP or 2 mM SNP for 24 h. The survivin and GAPDH levels were analyzed by the RT-PCR method. E, CL3 cells were treated with 1–2 mM SNP or 0.4–0.8 mM SNAP for 24 h. The total protein extracts were prepared for immunoblot analysis using anti-XIAP, Bcl-2, and ani-ERK-2 antibodies.

Article Snippet: Anti-phospho-p38 (9211S), anti-phosphocdc2-(Thr-161) (9114S), and anti-XIAP (2042) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Gene Expression, Western Blot, Incubation, Fluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer

doi: 10.1038/s41419-019-2001-7

Figure Lengend Snippet: a AGS (left) and MKN45 (right) cells treated with CBD for 24 h. Cells were collected for western blotting with the indicated antibodies. b AGS cells were treated with 4 μM of CBD for 24 h. Cells were immunostained with anti-XIAP (Red). Images were obtained using a confocal microscope. c , d AGS cells were transfected with myc-XIAP plasmid and treated with 4 μM CBD for 24 h. Apoptosis of cells was determined by flow cytometry ( c ) and immunoblotting ( d ). *** P < 0.001. e , f XIAP was knocked down using siRNA, treated with 4 μM CBD for 24 h, and subjected to flow cytometry ( e ) and west e rn blotting ( f )

Article Snippet: For RNA interference, XIAP siRNA (siXIAP), Smac siRNA (siSmac), and CHOP siRNA (siCHOP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Microscopy, Transfection, Plasmid Preparation, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer

doi: 10.1038/s41419-019-2001-7

Figure Lengend Snippet: a Cells were treated with CBD for 24 h. Total mRNA was extracted from cells and mRNA levels of XIAP were assessed by qRT-PCR. b Effect of CBD on XIAP protein stability was determined by performing cycloheximide chase assay. AGS cells were treated with 4 μM CBD and 50 μg/ml cycloheximide at indicated times. Cell lysate was analyzed by western blot analysis. c AGS cells were pre-treated with 5 μM of MG132 then treated with 4 μM of CBD for 24 h. Ubiquitination was detected by immunoblotting. β-Actin was used as a loading control . d 4 μM CBD was treated for various time periods with or without MG132 and analyzed by western blotting. e AGS cells were treated with 4 μM of CBD for 24 h. Cell lysates were immunoprecipitated with IgG or anti-Ub antibodies and immunoblotted for XIAP . f AGS cells were non-treated or pre-treated with 1 μM of MG132 for 1 h and then treated with 4 μM CBD for 24 h. Cell lysates were immunoprecipitated with IgG or anti-Ub antibodies and immunoblotted for XIAP. e The interaction between XIAP and Smac was assessed by Co-IP analysis

Article Snippet: For RNA interference, XIAP siRNA (siXIAP), Smac siRNA (siSmac), and CHOP siRNA (siCHOP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Western Blot, Ubiquitin Proteomics, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer

doi: 10.1038/s41419-019-2001-7

Figure Lengend Snippet: a Cells were treated with 4 μM (AGS cells) or 10 μM (MKN45 cells) of CBD for 24 h. Smac expression level was detected by western blotting analysis. b AGS cells were non-treated or pre-treated with 1 μM of MG132 for 1 h and then treated with 4 μM CBD for 24 h. The interaction between XIAP and Smac was determined by Co-IP analysis. c , d AGS cells were transfected with control siRNA (siNC) or Smac siRNA (siSmac). Transfected cells were treated with 4 μM of CBD for 24 h. The protein levels of Smac, XIAP ( c ), and apoptosis-related proteins ( d ) were detected by western blotting. e AGS cells were exposed to CBD for 24 h and then ER stress-related proteins were measured by western blotting. f The cells were transfected with siNC or CHOP-specific siRNA (siCHOP). The transfected cells were treated with 4 μM of CBD for 24 h. Cell lysates were immunoblotted with Smac antibody

Article Snippet: For RNA interference, XIAP siRNA (siXIAP), Smac siRNA (siSmac), and CHOP siRNA (siCHOP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Transfection, Control

Journal: Cell Death & Disease

Article Title: Cannabidiol promotes apoptosis via regulation of XIAP/Smac in gastric cancer

doi: 10.1038/s41419-019-2001-7

Figure Lengend Snippet: a Tumor growth after subcutaneous injection in an established in vivo tumor xenograft mouse model. ** P < 0.01. b Body weight of mice in the EtOH- and CBD-treated groups. c , d Representative images ( c ) and tumor weight ( d ) of tumor tissues from mice in the control and CBD treatment group. ** P < 0.01. e Tumor sections were stained with TUNEL dye using In Situ TUNEL detection kit. *** P < 0.001. f Tumor tissue sections were immunostained with an anti-XIAP antibody (Red)

Article Snippet: For RNA interference, XIAP siRNA (siXIAP), Smac siRNA (siSmac), and CHOP siRNA (siCHOP) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Injection, In Vivo, Control, Staining, TUNEL Assay, In Situ

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