full length recombinant human survivin his6  (R&D Systems)

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Details of antibodies used

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Concentration Assay

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Details of siRNA used

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques:

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Details of primers used for RT-PCR

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques:

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Survivin expression in human chondrosarcoma . Immunohistochemistry and immunoblot for survivin (red staining) from human chondrosarcoma specimens. A: Low-power image of human high-grade chondrosarcoma displays strong cellular expression of survivin protein. B: High-power magnification reveals the predominantly cytoplasmic staining, although strong nuclear signals are detectable. C and D: Other specimen of a grade III chondrosarcoma stained with monoclonal antibody, shows a similar pattern of staining. E: Strong survivin signal in a tumor cell displaying a mitotic figure (arrow). F: To verify the expression of survivin in human chondrosarcoma, immunoblots were performed from 3 high grade chondrosarcoma lysates (Patient Nr. 5, 7, 10). As control for the correct molecular weight, in vitro-transcribed and -translated (IVTT) recombinant survivin protein, derived from the full-length human cDNA was loaded. Furthermore, lysates from adult human cartilage served as a negative control. Total protein loaded was 1 μg for recombinant human survivin, 60 μg for chondrosarcoma and cartilage lysates. For A, B and E the polyclonal rabbit anti-survivin antibody AF886 was used. For C and D the monoclonal mouse anti-survivin antibody clone 32.1 was used. Original magnifications: 200× (A and C) and 400× (B and D) and 600× (E).

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Expressing, Immunohistochemistry, Western Blot, Staining, Control, Molecular Weight, In Vitro, Recombinant, Derivative Assay, Negative Control

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Survivin subcellular localization in human chondrosarcoma cells in vitro . Immunofluorescence localization of survivin using chondrosarcoma cells (SW1353) cultured on glass slides (A,D,G) and 4,6-diamidino-2-phenylindole-staining (DAPI) of the identical positions (B,E,H). Overlay of both stainings (C,F,I). A-C: The top row clearly shows the heterogeneous subcellular distribution from predominant cytoplasmic (lower cell) in the majority of the cell population to mixed cytoplasmic-nuclear in a smaller fraction of cells (upper cell). D-F: In a premitotic cell, survivin localizes to the mitotic spindle apparatus (arrow). Of note, here survivin signal appears stronger compared to the surrounding non-mitotic cells. G-I: In late telophase the mid-body (arrow) stains positive for survivin protein. Original magnifications: 400× (A-I)

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: In Vitro, Immunofluorescence, Cell Culture, Staining

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Suppression of survivin expression by transfection of siRNA . RNA interference was performed in SW1353 and Hs 819.T, either for GFP as control or for survivin. A: A pronounced decrease of survivin protein levels was measured by immunoblotting in SW1353 and Hs819.T. B: Quantitative real time PCR confirmed the subtotal suppression of survivin expression in SW1353 (left) and Hs819.T (right).

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Influence of survivin knockdown on proliferation and cell viability distribution of chondrosarcoma cells . The influences of RNA interference with survivin gene expression on cell viability and proliferation were measured by employing the MTT - assay (A) and by measuring BrdU incorporation (B). A: Cell viability was analyzed by MTT assay. Knockdown of survivin was performed at 0 hours and repeated at 48 hours in SW1353 (left) and Hs819.T (right). Significant reduction of viable cells compared to the untransfected control were seen in SW1353 after 48 hours and in Hs819.T at 72 hours.. Knockdown of GFP resulted in no significant alterations (Data not shown). The error bars represent +/-SEM. B: Proliferation of chondrosarcoma cell lines SW1353 and Hs819.T was measured by BrdU incorporation and subsequent detection employing a ELISA chemiluminescence immunoassay. Knock down was performed 24 hours prior to the incubation with BrdU. Knockdown of GFP resulted in no significant alterations. The error bars represent +/-SEM. A: Original results of one representative experiment are shown. B: Original results of three representative experiments are shown. P values less than 0.05 were considered significant (*).

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Knockdown, Gene Expression, MTT Assay, BrdU Incorporation Assay, Control, Enzyme-linked Immunosorbent Assay, Chemiluminescence Immunoassay, Incubation

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Effects of survivin knock down on cell cycle distribution in chondrosarcoma cells . SW1353 were transfected with siRNA targeting survivin and cell cycle distribution was determined by PI staining and FACS analysis after 24 hours. Both attached and detached cells were collected for the FACS analysis. GFP was transfected as control. Original dot blots and measured gates (left) and resulting histograms (right) are shown. The second peak of the resulting histogram represents the G2/M-phase fraction. The original results of one representative experiment are shown.

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Knockdown, Transfection, Staining, Control

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Influence of survivin knockdown on apoptotic rate of chondrosarcoma cells . Influences of RNA interference against survivin on programmed cell death of SW1353 (A and B) and Hs819.T (C and D) were measured by caspase 3/7 activity (A and C) and by analysing the sub-G 0/1 -phase fraction by using the fluorescence-activated cell sorting-propidium iodide staining method (B and D). Survivin knockdown resulted in moderate elevations of the indicators of apoptotic activity in SW 1353 (A and B, left) but not in Hs819.T (C and D). Transfection of GFP had no significant effects on apoptosis. Pronounced elevations of apoptotic markers were seen when the cells were stressed with doxorubicin 5 μM over 24 hours (A - D, right). The cytotoxic treatment resulted in a substantial increase of caspase 3/7 activity and fraction of apoptotic cells. Suppression of survivin sensitized the cells to doxorubicin treatment and further increased the apoptotic activity significantly in both cell lines. Again, transfection of GFP siRNA was used as a control. The error bars represent +/-SEM. P values less than 0.05 were considered significant (*). The original results of one representative experiment are shown.

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Knockdown, Activity Assay, Fluorescence, FACS, Staining, Transfection, Control

Journal: BMC Cancer

Article Title: The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

doi: 10.1186/1471-2407-11-120

Figure Lengend Snippet: Influence of survivin overexpression on proliferation and apoptosis of chondrosarcoma cells in vitro . A: Overexpression of human full length survivin by transfection of plasmid DNA led to a significant increase of protein level, as measured by immunoblot. Empty vector pcDNA3 was transfected as control. B: MTT - analysis over 5 days after transfection showed no influences on the proliferative activity of SW1353. C and D: Overexpression of survivin resulted in no alterations of spontaneous apoptotic rate as measured by caspase 3/7 activity (C) and by analysing the sub-G 0/1 -phase fraction by using the fluorescence-activated cell sorting-propidium iodide staining method (D). When SW1353 cells were exposed for 24 hours to doxorubicin (5 μM) (right) the apoptotic fraction and caspase activity of not transfected and pcDNA3 transfected cells increased markedly. Transfection of survivin resulted in significantly reduced rates of apoptosis after cytotoxic treatment. The error bars represent +/-SEM. P values less than 0.05 were considered significant (*). The original results of one representative experiment are shown.

Article Snippet: Recombinant full-length human survivin served as positive control (R&D Systems).

Techniques: Over Expression, In Vitro, Transfection, Plasmid Preparation, Western Blot, Control, Activity Assay, Fluorescence, FACS, Staining

Journal:

Article Title: The Tumor Gene Survivin Is Highly Expressed in Adult Renal Tubular Cells

doi: 10.2353/ajpath.2007.070132

Figure Lengend Snippet: Table 2

Article Snippet: Fifty μg of purified full-length recombinant human survivin peptide (R&D Systems) were incubated with 5 μl of primary rabbit anti-survivin antibody (AF886; R&D Systems) in 200 μl of PBS at 37°C for 2 hours.

Techniques: Expressing

Journal: European journal of immunology

Article Title: cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

doi: 10.1002/eji.200635943

Figure Lengend Snippet: Figure 2. Expression of IAP family members in freshly purified normal eosinophils and HES eosinophils compared to normal neutrophils. (A) Immunoblotting. Normal blood eosinophils (n = 2) and neutrophils (n = 2) expressed detectable levels of XIAP, but not cIAP-1, cIAP-2, NAIP, or survivin. In contrast, HES eosinophils (n = 4) expressed cIAP-2 and survivin. Filters were re-probed with anti-GAPDH mAb to ensure equal loading of the gels. As positive controls, recombinant survivin as well as Jurkat and HL-60 cell lysates were used (not shown). (B) RNase protection assay. Freshly isolated normal neutrophils and HES eosinophils were analyzed. cIAP-2 mRNA was strongly ex- pressed in HES eosinophils. Right side: Results of the densitometry analysis (as percentages) are presented (GAPDH expression was defined as 100% in each granulocyte popula- tion).

Article Snippet: Purified full-length recombinant human survivin (20 ng; R&D Systems) as well as lysates from Jurkat or HL-60 cells were used as positive controls.

Techniques: Expressing, Purification, Western Blot, Recombinant, Rnase Protection Assay, Isolation

Journal: European journal of immunology

Article Title: cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

doi: 10.1002/eji.200635943

Figure Lengend Snippet: Figure 3. Survivin and cIAP-2 expression in HES eosinophils under in vivo inflammatory conditions. Following immunos- taining, BAL cells and biopsies from HES patients were analyzed by confocal microscopy. Tissue eosinophils were specifically detected using an anti-ECP mAb. BAL cells were stained with PI to demonstrate the typical morphology of the nuclei of eosinophils. The bars represent 10 lm.

Article Snippet: Purified full-length recombinant human survivin (20 ng; R&D Systems) as well as lysates from Jurkat or HL-60 cells were used as positive controls.

Techniques: Expressing, In Vivo, Confocal Microscopy, Staining

Journal: European journal of immunology

Article Title: cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

doi: 10.1002/eji.200635943

Figure Lengend Snippet: Figure 4. Elevated survivin and cIAP-2 levels as well as reduced caspase-3 cleavage in normal eosinophils exposed to survival factors in vitro. (A) Eosinophils were stimulated with IL-3, IL-5, and GM-CSF for 3 h and analyzed by immunoblotting. The filter was re-probed with an anti-GAPDH mAb to ensure equal loading of the gel. (B) Spontaneous eosinophil apoptosis was associated with the occurrence of the 17-kDa caspase-3 cleavage product (22-h cultures are shown). Cleavage was accelerated in anti-Fas mAb-treated cells, whereas it was reduced in GM-CSF-stimulated eosinophils. Data are repre- sentative of four independent experiments.

Article Snippet: Purified full-length recombinant human survivin (20 ng; R&D Systems) as well as lysates from Jurkat or HL-60 cells were used as positive controls.

Techniques: In Vitro, Western Blot

Journal: European journal of immunology

Article Title: cIAP-2 and survivin contribute to cytokine-mediated delayed eosinophil apoptosis.

doi: 10.1002/eji.200635943

Figure Lengend Snippet: Figure 6. The cytochrome c-initiated caspase cascade in HES eosinophils can be accelerated by blocking survivin, XIAP, and cIAP-2. (A) Immunoblotting. HES eosinophil cell-free extract (derived from patient 1 in Table 1) was immunodepleted of survivin, XIAP, or cIAP-2. The IAP-depleted eosinophil extracts were analyzed regarding 20-lg/mL cytochrome c/dATP-inducible caspase-3 cleavage. The reactions were assembled at 37C for the indicated times. Note that depletion of each IAP resulted in accelerated caspase-3 cleavage. The presence of the caspase-3 fragments was quantified by densitometry analysis. A ratio between the sum of the fragments and the pro-caspase was calculated and is indicated below the immunoblots. (B) Caspase-3 activity assay. HES eosinophil cell-free extracts were immunodepleted of survivin, XIAP, or cIAP-2. The IAP-depleted eosinophil extracts were analyzed regarding 20-lg/mL cytochrome c/dATP-inducible caspase-3-like enzymatic activity. The reactions were assembled at 37C for 60 min. Note that depletion of each IAP resulted in accelerated caspase-3-like activity. Results were obtained using HES eosinophils from two different patients (experiments 1 and 2 corresponding to patients 8 and 9 in Table 1, respectively).

Article Snippet: Purified full-length recombinant human survivin (20 ng; R&D Systems) as well as lysates from Jurkat or HL-60 cells were used as positive controls.

Techniques: Blocking Assay, Western Blot, Derivative Assay, Caspase-3 Activity Assay, Activity 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: 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, 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: 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: Expressing, Real-time Polymerase Chain Reaction, Transfection, 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, Caspase-3 Activity Assay, Activity Assay