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: 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: BMB Reports

Article Title: The ADAM15 ectodomain is shed from secretory exosomes

doi: 10.5483/BMBRep.2015.48.5.161

Figure Lengend Snippet: The a disintegrin and metalloproteinase 15 (ADAM15) ectodomain is detected in the extracellular compartment. (A) Immunoblotting (IB) analysis of ADAM15-transfected HEK293F cells and isolated ADAM15 exosomes with antibody against the ADAM15 cytoplasmic domain (ADAM15 Cyto). HEK293F cells were transiently transfected with an empty or the ADAM15 expression vector and incubated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) in serum-free medium for 24 h. Cell lysates and isolated exosomes from conditioned medium (CM) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). P and M indicate the pro-form and mature-form of ADAM15, respectively. (B) Western blotting analysis of CM with antibody against the ADAM15extracellular (ADAM15 Ecto) or cytoplasmic domain (ADAM15 Cyto). ADAM15-transfected HEK293F cells were incubated with 5 ng/ml PMA in serum-free medium for 24 h. The CM was concentrated prior to SDS-PAGE.

Article Snippet: PMA (P1585) and the recombinant human ADAM15 ectodomain (WBC027) were purchased from SigmaAldrich (St. Louis, MO, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Western Blot, Transfection, Isolation, Expressing, Plasmid Preparation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: BMB Reports

Article Title: The ADAM15 ectodomain is shed from secretory exosomes

doi: 10.5483/BMBRep.2015.48.5.161

Figure Lengend Snippet: Ectodomain shedding of a disintegrin and metalloproteinase 15 (ADAM15) from exosomes. (A) ADAM15-rich exosomes were isolated from ADAM15-transfected 293F cells stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. The purified exosomes were further incubated at 37 ℃ for 10 h with or without conditioned medium from MDAH2774 ovarian cancer cells (CM-cancer). The samples were analyzed by immunoblotting with the indicated antibodies. (B) THP1 cells were induced to undergo monocytic differentiation with 20 ng/ml PMA for 24 h, and exosomes were isolated from CM of differentiated THP1 cells. Purified exosomes were further incubated as described above. The samples were analyzed by immunoblotting with the indicated antibodies. (C) Exosomes were purified and incubated with universal protease inhibitor (PI) as described above. The samples were analyzed by immunoblotting with the indicated antibodies.

Article Snippet: PMA (P1585) and the recombinant human ADAM15 ectodomain (WBC027) were purchased from SigmaAldrich (St. Louis, MO, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Isolation, Transfection, Purification, Incubation, Western Blot, Protease Inhibitor

Journal: BMB Reports

Article Title: The ADAM15 ectodomain is shed from secretory exosomes

doi: 10.5483/BMBRep.2015.48.5.161

Figure Lengend Snippet: Effect of specific protease inhibitor on a disintegrin and metalloproteinase 15 (ADAM15) shedding. (A, B) Purified exosomes were incubated in the presence or absence of 1 mM phenylmethylsulfonyl fluoride (PMSF) or ethylenediaminetetraacetic acid (EDTA). The samples were analyzed by immunoblotting with the indicated antibodies. #1 and #2 indicate sets of independent experiments (A). Tight panel represents the mean ± standard deviation of three independent experiments (B). ADAM15 shedding levels are expressed as signal intensity ratios (cytoplasmic fragment/full-length). P-values were calculated using the unpaired Student’s t -test.

Article Snippet: PMA (P1585) and the recombinant human ADAM15 ectodomain (WBC027) were purchased from SigmaAldrich (St. Louis, MO, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Protease Inhibitor, Purification, Incubation, Western Blot, Standard Deviation

Journal: BMB Reports

Article Title: The ADAM15 ectodomain is shed from secretory exosomes

doi: 10.5483/BMBRep.2015.48.5.161

Figure Lengend Snippet: A disintegrin and metalloproteinase 15 (ADAM15) ectodomain inhibits vitronectin-induced cell migration and MEK/extracellular regulated kinase (ERK) activation. (A) Effect of the ADAM15 ectodomain on vitronectin-induced cell migration. MDAH2774 cells were placed in an 8-㎛ pore-sized Transwell insert, and migration of the cells toward vitronectin (1.25 μg/ml) was analyzed in the presence of the recombinant ADAM15 ectodomain (rA15 ecto: 0.15 μM) or ADAM15-rich exosomes (A15 exo: 2 μg). After a 16 h incubation, the number of migrated cells in each field was determined by light microscopy (20 × original magnification) and ImageJ software. Lower panel represents the mean ± standard deviation (SD) of three independent experiments. (B) Effect of the ADAM15 ectodomain on vitronectin-induced activation of MEK/ERK signaling. MDAH2774 cells were cultured on vitronectin-coated plates (1.25 μg/ml) in the presence of the ADAM15 ectodomain (rA15 ecto: 0.15 μM) or ADAM15-rich exosomes (A15 exo: 2 μg) for 1 h. MEK or ERK phosphorylation was analyzed by immunoblotting (IB) with the indicated antibodies. Right panels represent mean ± SD of three independent experiments. Phosphorylation levels are expressed as signal intensity (fold-change relative to control). P-values were calculated using the unpaired Student’s t -test.

Article Snippet: PMA (P1585) and the recombinant human ADAM15 ectodomain (WBC027) were purchased from SigmaAldrich (St. Louis, MO, USA) and R&D Systems (Minneapolis, MN, USA), respectively.

Techniques: Migration, Activation Assay, Recombinant, Incubation, Light Microscopy, Software, Standard Deviation, Cell Culture, Phospho-proteomics, Western Blot, Control