Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Expression of IAP family members in freshly purified immature and mature neutrophils. (A) Immunoblotting. Mature neutrophils expressed detectable levels of XIAP, IAP-1, IAP-2, and survivin. NAIP was often not detectable. Immature neutrophils expressed large amounts of survivin. Filters were reprobed with anti-GAPDH or anti–β-actin mAbs to ensure equal loading of the gels. For both immature and mature neutrophil populations, results from three different donors are shown ( – ). (B) Confocal microscopy. Survivin was readily detected in immature, but not in mature, neutrophils. Survivin was localized in both nucleus and cytoplasm of immature cells. Caspase-3 was expressed in the cytoplasm of both immature and mature neutrophils. Interestingly, myeloblasts (white arrows) did not express detectable levels of caspase-3. Bars, 10 μm. The results are representative of five independent experiments.

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: Expressing, Purification, Western Blot, Confocal Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Survivin levels are markedly increased in mature neutrophils exposed to survival factors in vitro and in neutrophils from patients with CF. (A) Immunoblotting. Levels of survivin in neutrophils were increased by culturing normal control neutrophils in the presence of GM-CSF or G-CSF for 12 h. Moreover, freshly purified neutrophils from CF patients ( n = 4) showed strongly increased survivin protein levels compared with control blood neutrophils. 20 ng purified recombinant survivin served as a positive control. The filter was reprobed with an anti–β-actin mAb to ensure equal loading of the gel. (B) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Neutrophils from patients with CF contained increased amounts of survivin mRNA compared with normal neutrophils (left). The increase in survivin mRNA was mimicked by addition of GM-CSF or G-CSF to normal control neutrophils in vitro (right). Values are means ± SEM of three independent experiments. *, P < 0.05; ***, P < 0.001. (C) Proliferation assay. Freshly purified blood neutrophils from normal donors or CF patients did not demonstrate evidence for cell proliferation. The same was true for G-CSF– or GM-CSF–stimulated normal mature neutrophils. In contrast, immature neutrophils demonstrated significant proliferation, which was inducible by both GM-CSF and G-CSF. Pan–T cell–stimulated PBMCs were used as positive controls. Results of four independent experiments are shown for each cell population.

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: In Vitro, Western Blot, Control, Purification, Recombinant, Positive Control, Real-time Polymerase Chain Reaction, Proliferation Assay

Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Survivin expression in mature neutrophils under in vivo inflammatory conditions. (A) Immunohistochemistry. Cells morphologically representing neutrophils expressed survivin in tissue sections from patients with appendicitis. A control antibody did not stain the cells. (B). Confocal microscopy. Tissue neutrophils from patients with CF ( n = 3), ulcerative colitis ( n = 3), and appendicitis ( n = 3) demonstrated evidence for survivin expression. Neutrophils were specifically detected using an anti-CD15 mAb. Note that in ulcerative colitis and appendicitis, some neutrophils were survivin negative (red cells in the overlay panel). Bars, 10 μm.

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: Expressing, In Vivo, Immunohistochemistry, Control, Staining, Confocal Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Survivin is predominantly localized in the cytoplasm of mature CF neutrophils and associated with low caspase-3 enzymatic activity. (A) Confocal microscopy. Survivin was readily detected in CF blood neutrophils where it is mostly localized in the cytoplasm. Bars, 10 μm. Three independent experiments are presented. (B) Caspase-3 activity assay. Neutrophils were analyzed after a 10-h culture period. Caspase-3 activity was much lower in CF compared with control neutrophils. In addition, when normal neutrophils were cultured in the presence of GM-CSF or G-CSF, significantly lower caspase-3 activity levels were measured. Values are means ± SEM of eleven (C), three (CF), four (G-CSF), and two (GM-CSF) independent experiments. ***, P < 0.001.

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: Activity Assay, Confocal Microscopy, Caspase-3 Activity Assay, Control, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Antisense oligonucleotide treatment specifically prevents increases in survivin gene expression and antiapoptosis mediated by GM-CSF or G-CSF in mature neutrophils. (A) Real-time PCR. The lung cancer cell line A549 served as a standard (100%). Oligonucleotides (as and ms) had no effect on survivin mRNA expression in the absence of survival cytokines after a 4-h transfection period (top). Survivin-antisense (as), in contrast with mismatch control oligonucleotides (ms), prevented increases in survivin mRNA expression upon GM-CSF (middle) or G-CSF (bottom) stimulation in a dose-dependent manner. Values are means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Immunoblotting. Protein expression of survivin in normal neutrophils was not affected by incubation with as or ms (not depicted). However, survivin-as, in contrast with ms, partially (GM-CSF) or completely (G-CSF) prevented cytokine-induced increases in survivin expression. To demonstrate the specificity of the survivin-as effects, we also measured XIAP and Mcl-1 levels in the experiments using G-CSF. The filters were reprobed with an anti–β-actin mAb to ensure equal loading of the gels, and results of the densitometry analysis in terms of percentage are presented below the immunoblots. Results are representative of three independent experiments. (C) DNA fragmentation assay. Targeting survivin expression by survivin-as increased spontaneous apoptosis (left). GM-CSF (middle) and G-CSF (right) prevented apoptosis, and survivin-as blocked cytokine-mediated survival in a dose-dependent manner. Neutrophils were cultured for 10 h. Values are means ± SEM of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Viability assay. Neutrophils from survivin +/− mice demonstrated reduced IL-3–mediated survival. Neutrophils were isolated from blood and cultured in the presence of IL-3 for 24 h before viability was measured. No difference in spontaneous cell death was noticed between survivin +/+ and survivin +/− mice (not depicted). Values are means ± SEM of four independent experiments. *, P < 0.05.

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Western Blot, Incubation, DNA Fragmentation Assay, Cell Culture, Viability Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

doi: 10.1084/jem.20032033

Figure Lengend Snippet: Survivin-antisense treatment abolishes the inhibitory effect of survival cytokines on caspase-3 activation in mature neutrophils. (A) Immunoblotting. Neutrophils cultured in the presence of GM-CSF for 10 h maintained greater amounts of the caspase-3 proform and had decreased levels of the 17-kD fragment compared with untreated or oligonucleotide-treated cells. Survivin-antisense treatment (as), but not mismatch control oligonucleotides (ms), increased caspase-3 processing in the presence of GM-CSF in a dose-dependent manner. The same results were obtained in two additional experiments. (B) Caspase-3 activity assay. Increased enzymatic activity was detectable in neutrophils undergoing spontaneous apoptosis compared with GM-CSF– or G-CSF–treated cells. Survivin-as but not ms increased caspase-3–like enzymatic activity in the presence of GM-CSF or G-CSF in a dose-dependent manner. Results of two independent experiments are shown (circles, experiment 1; triangles, experiment 2).

Article Snippet: 20 ng of purified full-length recombinant human survivin (R&D Systems) was used as positive control for antisurvivin immunoblotting.

Techniques: Activation Assay, Western Blot, Cell Culture, Control, Caspase-3 Activity Assay, Activity 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) 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