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 and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing

Journal: The Journal of biological chemistry

Article Title: The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

doi: 10.1016/j.jbc.2023.103048

Figure Lengend Snippet: Figure 2. Inhibition of ADAMTS1 by TIMPs. A, inhibition of versicanase activity by TIMP family members. TIMP1, TIMP2, TIMP3, and TIMP4 (each at 500 nM) were incubated with ADAMTS1 (100 nM) for 1 h at 37 C before addition of V1-5GAG and digestion for 2 h. Following SDS-PAGE under reducing conditions (5% β-mercaptoethanol) and immunoblotting, FL V1-5GAG and versikine (VSK) were detected by the anti-Vc antibody. A representative immunoblot is shown (n = 2 independent experiments). B, inhibition of peptidolytic activity. TIMPs (each 25 nM) were incubated with a nominal con- centration of 25 nM ADAMTS1 for 1 h at 37 C before addition of the QF peptide substrate fluorescein-5(6)-carbonyl-Ala-Glu-Leu-Asn-Gly-Arg-Pro-Ile-Ser-Ile- Ala-Lys (5(6)-TAMRA) (3.5 μM) and digestion for 2 h. Following subtraction of the background (reactions not containing ADAMTS1), values were converted into percentage of ADAMTS1 activity in the absence of TIMPs and reported as average ± SD (n = 3, each point representing a technical replicate), p < 0.05 by Mann-Whitney test. C, titration of ADAMTS1 with TIMP3. TIMP3 (0–16 nM) was incubated with ADAMTS1 (20 nM nominal concentration) at 37 C for 1 h, and residual activity against the QF peptide FAM-AELNGRPISIAK-Tamra (3.5 μM) was determined. A representative titration curve is shown, each point representing a mean of two technical replicates. Final concentration of ADAMTS1 following titration was 10 nM. FL, full-length; No E, no enzyme; No I, no inhibitor; QF, Quenched-Fluorescent; TIMP, tissue inhibitor of metalloproteinase.

Article Snippet: Semiquantitative proteoglycan cleavage assays Purified V1-5GAG (100 nM) was digested with ADAMTS1, in TNC-B buffer at 37 C for 2 h. Where indicated, 500 μM recombinant human TIMP1, TIMP2, TIMP3, or TIMP4 (R&D Systems, Cat. n.: 970-TM, 971-TM, 973-TM, 974-TSF) were preincubated with 100 nM ADAMTS1 for 1 h at 37 C before digestion.

Techniques: Inhibition, Activity Assay, Incubation, SDS Page, Western Blot, MANN-WHITNEY, Titration, Concentration Assay

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 1. Effect of MMP9 deficiency on embryonic kidney mat- uration. Representative photographs of E11.5 (A and B) and E12.5 (C and D) kidneys from MMP9/ (A and C) and MMP9/ mice (B and D) stained with calbindin D-28K to delineate ureteric bud (arrow). Please note the defect in ureteric bud elongation and branching in MMP9/ embryonic kidney. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Staining

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 2. Effect of MMP9 deficiency on mouse body weight, kidney weight, and nephron number. (A and B) Body weight of newborn (A) and adult (B) MMP9/ and MMP9/ kidneys. (C and D) Kidney weight of newborn (C) and adult (D) MMP9/ and MMP9/ kidneys. Kidney weight and body weight were similar at birth, whereas they were significantly decreased in adult MMP9/ mice. (E and F) Nephron number in newborn (E) and adult (F) MMP9/ and MMP9/ kidneys. Values are mean SEM. ***P 0.001 and **P 0.01 versus MMP9/ mice.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 3. Effect of MMP9 deficiency on renal func- tion, kidney fibrosis, and kidney morphology. (A) Serum creatinine level of MMP9/ and MMP9/

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 4. Effect of MMP9 deficiency on ap- optosis ex vivo. (A and B) Representative photographs of E11.5 kidneys from MMP9/ (A) and MMP9/ mice (B) stained with calbindin D-28K to delineate ureteric bud in red and TUNEL method (apoptotic nuclei in green). (C) Percentage of apoptotic nuclei in E11.5 kidneys. Microphotographs were taken from 10 different MMP9/ and MMP9/ kidneys. (D) Concentration of ac- tive caspase-3 in E11.5 kidneys, n 50. Note that apoptosis assessed by different methods was increased in MMP9-deficient mesen- chyme. Values are mean SEM. ***P 0.001 versus MMP9/-injected mice. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Ex Vivo, Staining, TUNEL Assay, Concentration Assay, Injection

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 5. Effect of MMP9 deficiency on ap- optosis in vitro. (A and B) Representative pho- tographs of E11.5 kidneys from MMP9/ (A) and MMP9/ mice (B) grown for 48 h in culture. Ureteric bud is stained with calbindin D-28K (red) and apoptotic nuclei by the TUNEL method (green). (C) Percentage of ap- optotic nuclei in E11.5 kidneys grown for 48 h. (D) Genomic DNA extracted from E11.5 control (lane 1) and MMP9/ kidney (lane 2) grown for 48 h in culture. The migration pro- file in lane 2 is characteristic of apoptosis. Values are mean SEM. ***P 0.001 versus MMP9/ mice, n 20. Bars: (A and B) 250 and (E and F) 25 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: In Vitro, Staining, TUNEL Assay, Control, Migration

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 6. Effect of MMP9 deficiency on SCF (A and B) and c-kit (C and D) expression in E11.5 kidneys. (A) Representative SCF Western blot performed with 20 g of total proteins from MMP9/ (lane 1) and MMP9/ (lane 2) kidneys. (B) Quantitative analysis of five blots, using -actin as an internal control, showed a 50% increase of SCF protein expression in MMP9/ kidneys. (C) Representative Western blots detecting c-kit (lanes 1 and 2) and phospho c-kit (lanes 3 and 4) in 20 g total proteins extracts from MMP9/ (lanes 1 and 3) and MMP9/ (lanes 2 and 4) kidneys. (D) Phospho c-kit expression related to -actin expres- sion. Note that c-kit expression is not modified by MMP9 defi- ciency, whereas phospho c-kit expression is reduced by 60% in MMP9/ kidneys. Values are mean SEM. ***P 0.001 versus MMP9/ mice.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Expressing, Western Blot, Control, Modification

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 7. Sensitivity of membrane SCF to MMP9 proteolysis in vitro. (A) Representative SCF Western blot performed with con- centrated medium obtained from 10 E11.5 MMP9/ (lane 1) or MMP9/ (lane 2) kidneys grown for 24 h. A band corresponding to the soluble form of SCF is observed in concentrated media from MMP9/ kidney only. (B) ELISA of sSCF in the culture medium of E11.5 MMP9/ (white bar) or MMP9/ (gray bar) kidneys grown for 24, 48, and 72 h. Values are mean SEM. ***P 0.001 versus MMP9/ mice, n 20.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Membrane, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 8. Effect of sSCF on apoptosis in vitro. Representative photographs of E11.5 kidneys from MMP9/ (A through F) and MMP9/ (G through L) kidneys stained with calbindin D-28K to delineate ureteric bud in red and the TUNEL method (apoptotic nuclei in green). Kidneys were grown for 48 h in the absence (A through C and G through I) or the presence (D through F and J through L) of physiologic concentration of sSCF in culture medium. sSCF in culture medium decreased the number of apoptotic cells and increased ureteric bud branching of MMP9/ (J through L versus G through I) and MMP9/ (D through F versus A through C) kidneys. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: In Vitro, Staining, TUNEL Assay, Concentration Assay

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 9. Effect of sSCF on E11.5 MMP9/ and MMP9/

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

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