Journal: International journal of oncology

Article Title: Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.

doi: 10.3892/ijo_00000464

Figure Lengend Snippet: Figure 1. Real-time RT-PCR quantitation of expression of survivin, HBXIP, XIAP and ß-actin mRNAs in non-small cell lung carcinoma tissues and matched lungs. The set of PCR amplification plots, representing the two-step (A-E, F-J and P-T) and the coupled (K-O) real-time RT-PCR assays with an input of 200 ng of total RNA and running in duplicate, shows the expression of survivin, HBXIP, XIAP and ß-actin mRNAs in five tumour (Tu)-lung (Lu) matched pairs. The tumours were: squamous cell lung carcinoma (SQCLC), lung adenocarcinoma (LAC), large-cell lung carcinoma (LCLC), sarcomatoid lung carcinoma (SLC), and undifferentiated lung carcinoma (UNDIF). NTC, no template control. ΔFU, background-subtracted fluorescence intensity (in arbitrary units) of the released reporter dye (6-FAM or VIC).

Article Snippet: The levels of survivin and XIAP proteins in cell and tissue extracts were measured by chromogenic sandwich enzyme-linked immunosorbent assays (ELISAs) using Human Survivin ELISA DuoSet IC (cat. no. DYC647) and Human XIAP ELISA DuoSet IC (cat. no. DYC822) kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Quantitation Assay, Expressing, Amplification, Control, Fluorescence

Journal: International journal of oncology

Article Title: Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.

doi: 10.3892/ijo_00000464

Figure Lengend Snippet: Figure 2. Analysis of survivin and XIAP expression in non-small cell lung carcinoma cell lines. (A and D) Expression of survivin and XIAP mRNAs in the tumour cell lines as quantitated by real-time RT-PCR. Data are represented as the mean ± standard error of the mean of three independent experiments. At the bottom of each column, the genotype at nucleotide -31 in the survivin gene (BIRC5) promoter is shown. (B and E) Expression of survivin and XIAP proteins in the tumour cell lines as analysed by SDS-PAGE and immunoblotting. (C and F) Correlation analysis of mRNA and protein expression for survivin and XIAP, respectively, in the tumour cell lines. Pearson linear correlation coefficients r and their P-value are indicated.

Article Snippet: The levels of survivin and XIAP proteins in cell and tissue extracts were measured by chromogenic sandwich enzyme-linked immunosorbent assays (ELISAs) using Human Survivin ELISA DuoSet IC (cat. no. DYC647) and Human XIAP ELISA DuoSet IC (cat. no. DYC822) kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot

Journal: International journal of oncology

Article Title: Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.

doi: 10.3892/ijo_00000464

Figure Lengend Snippet: Figure 3. Comparison of survivin mRNA (A) and XIAP mRNA (B) expression, respectively, in non-small cell lung carcinoma (NSCLC) cell lines and small cell lung carcinoma (SCLC) cell lines. In the box plot, the upper and the lower boundary of the box and the line within the box indicate the 75th and 25th percentiles and the median, respectively. The error bars above and below the box indicate the 90th and 10th percentiles. Statistical difference (P) between the transcript expression levels was calculated using the Mann-Whitney test.

Article Snippet: The levels of survivin and XIAP proteins in cell and tissue extracts were measured by chromogenic sandwich enzyme-linked immunosorbent assays (ELISAs) using Human Survivin ELISA DuoSet IC (cat. no. DYC647) and Human XIAP ELISA DuoSet IC (cat. no. DYC822) kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Comparison, Expressing, MANN-WHITNEY

Journal: International journal of oncology

Article Title: Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma.

doi: 10.3892/ijo_00000464

Figure Lengend Snippet: Figure 6. Correlation analysis of XIAP mRNA and protein expression in non-small cell lung carcinoma (NSCLC) tissues (A) and lungs (B). Pearson linear correlation coefficients r and their P-value are indicated.

Article Snippet: The levels of survivin and XIAP proteins in cell and tissue extracts were measured by chromogenic sandwich enzyme-linked immunosorbent assays (ELISAs) using Human Survivin ELISA DuoSet IC (cat. no. DYC647) and Human XIAP ELISA DuoSet IC (cat. no. DYC822) kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Expressing

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: 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: Cell Death & Disease

Article Title: Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes

doi: 10.1038/s41419-018-0508-y

Figure Lengend Snippet: a MEFs expressing mCherry-GFP-LC3b were transfected with siRNA against either cIAP1, cIAP2 or XIAP. Cells were analysed with the microscope and the number of mCherry+, and GFP + puncta/cell were calculated and the ratio of GFP + /mCherry + puncta is indicated. Shown are the means and the error bars represent the SEM of at least three independent experiments. Westerns show efficient knockdown of cIAP1 and XIAP expression. cIAP2 siRNA efficiency was determined by real time PCR as shown in the graph below the westerns. b Wild type and cIAP2 −/− XIAP −/− dermal fibroblasts expressing mCherry-GFP-LC3b were treated analysed on the microscope and mCherry+, and GFP + puncta/cell were calculated. The ratio of GFP+/mCherry + puncta is indicated. c Wild type and cIAP2 −/− XIAP −/− dermal fibroblasts were left in complete media (CM) or starved for 2 h in EBSS. Cells were lysed and proteins analysed by western blot for XIAP, LC3, and Actin. cIAP2 −/− was confirmed by PCR due to lack of effective antibodies for mouse cIAP2 (see supplemental Fig. ). d Immunofluoresence showing accumulation of LC3 in starved cIAP2 −/− XIAP −/− dermal fibroblasts. Wild type and cIAP2 −/− XIAP −/− dermal fibroblasts were incubated in complete media or starved in EBSS for 2 h, then fixed and stained for LC3 (green channel) and LAMP2 (red channel). Nuclei are stained blue with Hoechst. Shown in upper panels are overviews. Lower panels show zoomed in regions indicated in the upper panels

Article Snippet: The following antibodies were used; EGFR (PA1-1110—Thermo fisher), XIAP (MAB822—R&D Systems), cIAP1 (was a kind gift from John Silke—WEHI Melbourne Australia), LC3b (nb100–2220—Novus), LAMP2 (ab13524—Abcam).

Techniques: Expressing, Transfection, Microscopy, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Incubation, 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: 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