Journal: Arthritis Research & Therapy

Article Title: Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

doi: 10.1186/ar2603

Figure Lengend Snippet: Semiquantitative analysis scoring for TRAIL, TRAIL receptors, TUNEL, cleaved caspase-3, survivin and xIAP

Article Snippet: Recombinant human TRAIL (375-TL/CF) and monoclonal antibodies directed against TRAIL (MAB687, immunoglobulin (Ig) G1), TRAIL receptor 3/DcR1 (M430, IgG1), rabbit anti-human survivin (AF886, polyclonal IgG) and anti-human x-linked inhibitor of apoptosis protein (xIAP) (MAB8221, IgG1) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: TUNEL Assay

Journal: Arthritis Research & Therapy

Article Title: Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

doi: 10.1186/ar2603

Figure Lengend Snippet: TRAIL, TRAIL R1, TRAIL R2, TRAIL R3 and TRAIL R4 expression pattern. Positive cells are shown in red staining. Staining pattern in active RA synovial tissue demonstrated in the area indicated by arrows. Magnification × 200. Synovial tissues were obtained from normal (1st row), active rheumatoid arthritis (RA) (2nd row), inactive RA (3rd row), osteoarthritis (OA) (4th row) and spondyloarthropathy (SpA) patients (5th row). TRAIL = tumour necrosis factor related apoptosis inducing ligand. Regions of interest in some panels are indicated by the circles and arrows and discussed in the text.

Article Snippet: Recombinant human TRAIL (375-TL/CF) and monoclonal antibodies directed against TRAIL (MAB687, immunoglobulin (Ig) G1), TRAIL receptor 3/DcR1 (M430, IgG1), rabbit anti-human survivin (AF886, polyclonal IgG) and anti-human x-linked inhibitor of apoptosis protein (xIAP) (MAB8221, IgG1) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Staining

Journal: Arthritis Research & Therapy

Article Title: Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

doi: 10.1186/ar2603

Figure Lengend Snippet: TUNEL, cleaved caspase-3, survivin and xIAP protein expression (red) in five patient groups. Magnification ×200, except for x-linked inhibitor of apoptosis protein (xIAP) × 400. OA = osteoarthritis; RA = rheumatoid arthritis; SpA = spondyloarthropathies; TUNEL = terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling.

Article Snippet: Recombinant human TRAIL (375-TL/CF) and monoclonal antibodies directed against TRAIL (MAB687, immunoglobulin (Ig) G1), TRAIL receptor 3/DcR1 (M430, IgG1), rabbit anti-human survivin (AF886, polyclonal IgG) and anti-human x-linked inhibitor of apoptosis protein (xIAP) (MAB8221, IgG1) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: TUNEL Assay, Expressing

Journal: Arthritis Research & Therapy

Article Title: Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

doi: 10.1186/ar2603

Figure Lengend Snippet: Double labelling of active RA tissues for expression of (a) TRAIL (red) and CD68 (blue); (b) TRAIL R1 (red) and CD68 (blue); (c) TRAIL R4 (red) and CD68 (blue); (d) TRAIL (red) and CD3 (blue); (e) TRAIL R1 (red) and CD3 (blue); (f) TRAIL R4 (red) and CD3 (blue). An arrow indicates from where the magnified image at the bottom right corner of panel was obtained in panels a-c. Panel g shows double labelling of cleaved caspase-3 and xIAP. Panel h is a magnified region of the image that is indicated by the arrow and circle in panel g. Magnification of panels is × 200 and magnified areas are approximately × 600.

Article Snippet: Recombinant human TRAIL (375-TL/CF) and monoclonal antibodies directed against TRAIL (MAB687, immunoglobulin (Ig) G1), TRAIL receptor 3/DcR1 (M430, IgG1), rabbit anti-human survivin (AF886, polyclonal IgG) and anti-human x-linked inhibitor of apoptosis protein (xIAP) (MAB8221, IgG1) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium

doi: 10.1186/ar2603

Figure Lengend Snippet: Expression levels of caspase-3, survivin and xIAP mRNAs in inactive RA, active RA and OA patients. These levels were determined by real-time PCR. The results are normalised to levels of hARP and have been expressed relative to OA control tissue. *p < 0.05, compared with inactive RA. n = 3 (OA), n = 3 (inactive RA), n = 5 (active RA). OA = osteoarthritis; PCR = polymerase chain reaction; RA = rheumatoid arthritis; xIAP = x-linked inhibitor of apoptosis protein.

Article Snippet: Recombinant human TRAIL (375-TL/CF) and monoclonal antibodies directed against TRAIL (MAB687, immunoglobulin (Ig) G1), TRAIL receptor 3/DcR1 (M430, IgG1), rabbit anti-human survivin (AF886, polyclonal IgG) and anti-human x-linked inhibitor of apoptosis protein (xIAP) (MAB8221, IgG1) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Polymerase Chain Reaction

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:

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: 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