Journal: Cell Reports Medicine

Article Title: Vaccine-induced ICOS + CD38 + circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways

doi: 10.1016/j.xcrm.2021.100262

Figure Lengend Snippet:

Article Snippet: Anti-human Survivin (REA459) , Miltenyi , Cat # 130-106-741; RRID: AB_2653606.

Techniques: Recombinant, Staining, Gene Expression, Software

Journal: Scientific reports

Article Title: The proto-oncogene survivin splice variant 2B is induced by PDGF and leads to cell proliferation in rheumatoid arthritis fibroblast-like synoviocytes.

doi: 10.1038/srep09795

Figure Lengend Snippet: Figure 1. The survivin expression in blood and synovial tissues from RA and OA patients. (a) The serum survivin expression levels were analyzed using an ELISA. (RA, 22 cases; controls, 16 cases) (mean RA, 19.5 pg/ml vs. controls, 7.7 pg/ml; p = 0.044). An age- and sex-matched control group consisted of OA and healthy controls (RA, 58.8 ± 15.6 years old; controls, 53.9 ± 19.8 years old; p = 0.404). t-test, *p < 0.05. (b) The survivin expressions in RA and OA synovial tissues were analyzed using IHC (survivin: red, upper sections x40, lower sections x20). The data shown are representative of the samples. Arrows indicate the endothelium of synovia. (c–f) Dual staining in immunohistochemistry for RA synovial tissues. Red pigment and arrows indicate the expression of each lineage marker (CD55; a marker of FLS, CD4; a marker of CD4+ T lymphocytes, CD8; a marker of CD8+ T lymphocytes, MPO (myeloperoxidase); a marker of neutrophil). Green pigment indicates survivin. (x40)

Article Snippet: The serum survivin levels were measured with a Quantikine ELISA kit for human survivin (R&D systems) according to the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Staining, Immunohistochemistry, Marker

Journal: Scientific reports

Article Title: The proto-oncogene survivin splice variant 2B is induced by PDGF and leads to cell proliferation in rheumatoid arthritis fibroblast-like synoviocytes.

doi: 10.1038/srep09795

Figure Lengend Snippet: Figure 6. The serum expression levels of survivin-2B in RA patients determined using an ELISA. (a) We collected serum samples from 35 RA patients and 21 age- and sex- matched controls (healthy controls and OA patients), and measured the serum survivin-2B levels with an ELISA (mean RA, 18.7 ng/ml vs. controls, 4.7 ng/ml; p = 0.049) (RA, 64.7 ± 11.4 years old; controls, 59.6 ± 18.0 years old; p = 0.250). The gray lines indicate the median of survivin-2B level in each group. (b) We compared the DAS28-ESR between the survivin-2B high RA group (n = 18) and the survivin-2B low RA group (n = 21) (mean RA, 4.64 vs. controls, 3.64; p = 0.037). t-test, ∗p < 0.05.

Article Snippet: The serum survivin levels were measured with a Quantikine ELISA kit for human survivin (R&D systems) according to the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent 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, 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: International Journal of Molecular Sciences

Article Title: Arachidin-1, a Prenylated Stilbenoid from Peanut, Induces Apoptosis in Triple-Negative Breast Cancer Cells

doi: 10.3390/ijms23031139

Figure Lengend Snippet: Western blot analysis and densitometry measurements of treated TNBC cell lines with arachidin-1 (A-1, 0.1, and 1 µM). Western blot images of ( A ) PARP and cleaved PARP, GAPDH loading control. ( B ) Full-length caspase-8, GAPDH loading control; ( C ) cleaved caspase-9, vinculin loading control; and ( D ) survivin, GAPDH loading control. Lysates (60 µg of total protein) of MDA-MB-231 and MDA-MB-436 cells treated with A-1 were analyzed by western blotting. The relative densitometry of ( E ) full-length PARP, ( F ) cleaved PARP, ( G ) caspase-8, ( H ) cleaved caspase-9, and ( I ) survivin protein levels were analyzed by comparing to the loading control. Cells with 0.01% DMSO were used as controls. Data represents mean ± SD from three or more independent experiments. * p < 0.05, ** p < 0.001, **** p < 0.0001 versus control in MDA-MB-231, # p < 0.05, ## p < 0.001, #### p < 0.00001 versus control in MDA-MB-436, and ns = not significant from control.

Article Snippet: The primary antibodies and dilutions included rabbit anti-caspase-8 (D35G2) mAb (1:500; Cell Signaling Technology; Danvers, MA, USA), rabbit anti-PARP mAb (1:500; Cell Signaling Technology; Danvers, MA, USA), rabbit anti-human survivin polyclonal antibody (0.5 μg/mL; R&D Systems; Minneapolis, MN, USA), mouse anti-human caspase-9 mAb (1 μg/mL; R&D Systems; Minneapolis, MN, USA), mouse anti-vinculin antibody (2 μg/mL; R&D Systems; Minneapolis, MN, USA), and mouse anti-human GAPDH antibody (0.05 μg/mL; R&D Systems; Minneapolis, MN, USA).

Techniques: Western Blot, Control