Journal: BBA Clinical

Article Title: Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients

doi: 10.1016/j.bbacli.2017.05.003

Figure Lengend Snippet: Label-free mass spectrometry data. List of statistically significant discovered proteins using LC-MS analysis. Proteins (in bold) were selected for further validation using ELISAs.

Article Snippet: Four commercially available kits for these four proteins; angiogenin (ANG) [Abcam, UK - ab99970], clusterin (CLU) [R&D system, UK - DCLU00], C-C Motif Chemokine 18 (CCL18) [Abcam, UK - ab100620], and Complement C1q [Abcam, UK - ab170246] were used.

Techniques: Mass Spectrometry, Biomarker Discovery, Variant Assay

Journal: BBA Clinical

Article Title: Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients

doi: 10.1016/j.bbacli.2017.05.003

Figure Lengend Snippet: ELISA Data. Mean, SD, Area under the curve (AUC) and p -value for each of the new and standard proteins found in the two groups of patients compared.

Article Snippet: Four commercially available kits for these four proteins; angiogenin (ANG) [Abcam, UK - ab99970], clusterin (CLU) [R&D system, UK - DCLU00], C-C Motif Chemokine 18 (CCL18) [Abcam, UK - ab100620], and Complement C1q [Abcam, UK - ab170246] were used.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: BBA Clinical

Article Title: Novel panel of protein biomarkers to predict response to bortezomib-containing induction regimens in multiple myeloma patients

doi: 10.1016/j.bbacli.2017.05.003

Figure Lengend Snippet: Logistic regression analysis data. List of different protein combinations used to establish the best model that can be used as a predictive panel for response to induction therapy containing bortezomib regime.

Article Snippet: Four commercially available kits for these four proteins; angiogenin (ANG) [Abcam, UK - ab99970], clusterin (CLU) [R&D system, UK - DCLU00], C-C Motif Chemokine 18 (CCL18) [Abcam, UK - ab100620], and Complement C1q [Abcam, UK - ab170246] were used.

Techniques:

Journal: Proteomics

Article Title: Identification, prioritization and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody-based assays on tissue specimens

doi: 10.1002/pmic.201200541

Figure Lengend Snippet: Standard Curve of ELISA assays for protein quantification of cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP-1) and cathepsin L. A), dose-response curves for COMP. B), dose-response curve for periostin. C), dose-response curve for VAP-1. D), dose-response curve for cathepsin L.

Article Snippet: Materials Hydrazide resin and sodium periodate were from Bio-Rad (Hercules, CA); sequencing-grade trypsin and TMB reagent were from Promega (Madison, WI); PNGase F was from New England Biolabs (Ipswich, MA); C18 columns were from Waters (Milford, MA); Recombinant protein, capture and detection antibody of human cartilage oligomeric matrix protein (COMP), periostin, cathepsin L, clusterin and galectin-3 binding protein, streptavidin-HRP conjugates, ELISA plates and human VAP-1 Quantikine ELISA Kit were from R&D systems (Minneapolis, MN); All other chemicals were from Sigma-Aldrich (St. Louis, MO).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 3. Clusterin in human pituitary adenomas. Confocal image of A) human pituitary adenoma and non-tumorous pituitary tissue specimens showing clusterin (green) expressed exclusively in the cytoplasm; B) Co-localization of clusterin with GH, PRL and aGSU in respective human pituitary adenoma specimens (clusterin green, respective hormones red). doi:10.1371/journal.pone.0017924.g003

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques:

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 4. Pituitary proliferation, DNA damage and senescence markers in the aGSU.PTTG pituitary gland. A) In vivo BrdU incorporation. Mice were injected with BrdU (50 mg/g BW), and pituitary sections stained for BrdU. One thousand cells/section, 3 sections/animal, n = 3 animals/ genotype were analyzed. *, p,0.05; Western blot analysis of B) proliferation markers; C) DNA damage, DNA repair and p53-dependent senescence markers, and D) oncogene-induced senescence markers; E) Confocal image showing immunofluorescent cytoplasmic clusterin, and intranuclear p15 and p16 expression (green) in WT and in pre-tumorous aGSU.PTTG pituitary glands, and in aGSU.PTTG pituitary adenomas; F) Pituitary SA-b- galactosidase enzymatic activity (blue) in WT and in pre-tumorous aGSU.PTTG pituitary gland. Three pituitary cryosections/animal were analyzed from 3 animals/genotype, and a representative image shown. Western blots here and elsewhere were repeated 3 times with similar results and representative blots shown. doi:10.1371/journal.pone.0017924.g004

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: In Vivo, BrdU Incorporation Assay, Injection, Staining, Western Blot, Expressing, Activity Assay

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 6. C/EBPs induce clusterin. A) C/EBPb is up-regulated in the aGSU.PTTG pituitary. Confocal image showing C/EBPb co-localization with aGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous aGSU.PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPb–red, intranuclear); B) Western blot analysis of C/EBPb and d isoforms induced in LbT2 cells stably transfected with mPttg; C) Effects of C/EBPs on the clusterin promoter in LbT2 and aT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and 800 ng murine pCDNA3-C/EBPa, b or d. The ratio of luciferase to co-trasfected b-galactosidase control reporter vector was normalized to pCDNA3- null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown.*, p,0.05, **,p,0.01; D) Western blot analysis of clusterin expression in gonadotroph-derived aT3 cells 24 hours after transfection with pCDNA3-C/EBPb or E) pCDNA3-C/EBPd; F) Western blot analysis of clusterin expression in LbT2 mPttg cells 48 hours after simultaneous transfection with siC/EBPb and siC/EBPd (3 nM each). Two different combinations of siRNAs were used. doi:10.1371/journal.pone.0017924.g006

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Control, Expressing, Derivative Assay

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 7. Clusterin restrains pituitary cell proliferation by inducing Cdk inhibitors. Western blot analysis of Cdk inhibitors and proliferation markers A) in LbT2 cells, B) in aT3 cells 48 h after transfection with mClu; C) Confocal images of immunofluoprescence of histone H3 methylation on lysine 9 (H3-K9M) (red) in vector and Clu-expressing aT3 cells 48 hours after transfection; D) Quantification of positive H3-K9M foci. Cells were fixed, stained with H3-K9M antibody, and one thousand cells/field counted in three randomly chosen visual fields; E) Percentage of BrdU positive cells 48 h after transfection with mClu. Triplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry, *, p,0.05; F) aT3 cells stably overexpressing mClu or vector were synchronized in 0.1% fetal bovine serum for 18 hours, and then cultured in 10% fetal bovine serum. At the indicated times, duplicate samples were pulsed with BrdU for 30 min, analyzed by flow cytometry, and cells in S-phase identified by staining with BrdU antibodies. doi:10.1371/journal.pone.0017924.g007

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Transfection, Methylation, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Stable Transfection, Cell Culture

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 8. Clusterin attenuation promotes proliferation. Western blot analysis of Cdk inhibitors and proliferation markers A) in LbT2 cells, B) in aT3 cells; C) Percentage of BrdU positive cells 48 h after transfection with siClu. D) Upper panel, Western blot confirms p15 down-regulation, Lower panel, Percentage of BrdU positive LbT2 cells 48 h after transfection with sip15. E) Upper panel, Western blot confirms p16 down-regulation, Lower panel, Percentage of BrdU positive LbT2 cells 48 h after transfection with sip16. For BrdU detection, cells were fixed, stained with BrdU antibody and one thousand cells/field in three randomly chosen fields counted. *, p,0.05. doi:10.1371/journal.pone.0017924.g008

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Transfection, Staining

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 9. FOXL2 stimulates the clusterin promoter. A) Effects of FOXL2 on the clusterin promoter in aT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co- trasfected b-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown; **,p,0.01; B) Western blot analysis of clusterin expression in aT3 cells 24 hours after transfection with pcDNA3-His-mFoxl2; C) ChiP assay was performed in nuclear fractions derived from aT3 cell lysates. Top, schematic of the approximate location of primers used in the PCR reactions. Enrichment of specific clusterin promoter sequences was obtained with primer Set 2. FOXL2, specific antibody, IgG, nonspecific antibody, PCP, positive control primers. The experiment was repeated twice, and results of a representative assay shown. doi:10.1371/journal.pone.0017924.g009

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Transfection, Plasmid Preparation, Luciferase, Control, Expressing, Western Blot, Derivative Assay, Positive Control

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 10. Proliferation restricting pathways in the pituitary gonadotroph cell lineage. FOXL2 directly activates the clusterin promoter, while Pttg overexpression results in proliferation, DNA damage and stimulation of C/EBPb and d; C/EBPs activate the clusterin promoter. High levels of secretory clusterin trigger expression of Cdk inhibitors p15, p16 and p27, and C/EBPb also cooperates to induce p15. Up-regulated tumor suppressor proteins likely underlie proliferation restraint preventing uncontrolled growth of benign pituitary adenomas of gonadotroph cell origin. doi:10.1371/journal.pone.0017924.g010

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Over Expression, Expressing

Journal: OncoImmunology

Article Title: Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

doi: 10.1080/2162402x.2019.1629257

Figure Lengend Snippet: Figure 1. Clusterin expression in luminal breast cancer samples. A and B. Clusterin expression was analyzed by immunohistochemistry in tumoral (a) and juxtatumoral (b) tissues. Representative pictures are shown (n = 3, bars: 50 µm). C. Clusterin expression was quantified in tumor samples and their juxtatumoral counterparts by ELISA. Results are expressed as the amount of clusterin/total protein (µg of clusterin/mg of total protein, n = 21, ns = no statistically significant differences, p = .31).

Article Snippet: Total clusterin was detected by ELISA using a conventional ELISA kit (Human Clusterin DuoSet ELISA, R&D Systems).

Techniques: Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

doi: 10.1080/2162402x.2019.1629257

Figure Lengend Snippet: Figure 2. Fucosylated clusterin (fCLU) is expressed in luminal breast cancer and interacts with DC-SIGN. a. Scheme of the ELISA assay used to detect fucosylated clusterin. Clusterin is captured by a mAb directed to clusterin and the presence of fucosylated clusterin (fCLU) is revealed by using either biotinilated Ulex europaeus agglutinin-I lectin (UEA-I) or Lotus tetragonolobus lectin (LT). b. Fucosylated semen clusterin, but not serum or recombinant clusterin (blue bars), was detected by the assay described in (a). The addition of α-L-fucose prevents the recognition of fucosylated semen clusterin (red bars). Results are expressed as the mean ± SD of 4 independent experiments. c and d. Fucosylated clusterin expression in breast tumor and juxtatumoral samples (n = 14–21) was analyzed using Lotus tetragonolobus lectin (c) or Ulex europaeus agglutinin-I (d). Results are expressed as the amount of fucosylated clusterin relative to the amount of total clusterin (left panels) or total protein (right panels) (***p < .001, **p < .01, *p < .05). E. The ability of clusterin from tumor samples and juxtatumoral samples to bind to DC-SIGN was evaluated. Upper panel: the presence of clusterin was evaluated using a mAb directed to clusterin. Lower panel: the membrane was stripped and revealed using DC-SIGN-huFc and HRP conjugated anti-huIgG. Of note, total clusterin but not total protein was used to normalize the amount of sample loaded into the gel for each tumor and juxtatumor sample pair. Accordingly, no loading control was analyzed. Panel F shows the ratio between DC-SIGN-huFC binding and total CLU. The fold change between tumor and juxtatumor CLU ratios are shown.

Article Snippet: Total clusterin was detected by ELISA using a conventional ELISA kit (Human Clusterin DuoSet ELISA, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Membrane, Control, Binding Assay

Journal: OncoImmunology

Article Title: Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

doi: 10.1080/2162402x.2019.1629257

Figure Lengend Snippet: Figure 6. Fucosylated clusterin promotes the differentiation of macrophages into a proangiogenic profile. a-c. Macrophages were differentiated from monocytes cultured with M-CSF for 5 days, in the absence or presence of semen fucosylated clusterin. Then, cells were stimulated, or not, with LPS (10 ng/ml), and the pattern of cell clustering (a), the expression of HLA-DR, CD86, PDL-1, CD40 and CD80 (b), and the production of VEGF, IL-8, TNF-α, IL-10, and IL-6 (c) were evaluated by optical microscopy, flow cytometry or ELISA. Representative pictures (bars = 100µm) and histograms (n = 3–9) are shown in a and b. On b, the control condition mean fluorescent intensities (MFI) of each independent experiment were normalized as 1. The mean of 5–9 independent experiments is shown. The mean of 5–7 independent experiments is shown in c (**p < .01, *p < .05).

Article Snippet: Total clusterin was detected by ELISA using a conventional ELISA kit (Human Clusterin DuoSet ELISA, R&D Systems).

Techniques: Cell Culture, Expressing, Microscopy, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: OncoImmunology

Article Title: Aberrant fucosylation enables breast cancer clusterin to interact with dendritic cell-specific ICAM-grabbing non-integrin (DC-SIGN)

doi: 10.1080/2162402x.2019.1629257

Figure Lengend Snippet: Figure 5. Fucosylated clusterin interacts with macrophages. a. Expression of DC-SIGN by monocyte-derived macrophages. A representative experiment (n = 7) is shown. b. Macrophages were incubated for 30 min at 4°C with different concentrations of fucosylated clusterin isolated from semen. Cells were then washed, lysed and the binding of clusterin was revealed by ELISA. Results are expressed as the mean ± SD of 4 independent experiments. c. Macrophages were incubated for 30 min at 4°C with fucosylated clusterin (10 µg/ml), in the absence or presence of a neutralizing mAb directed to DC- SIGN (5 µg/ml), lactose (100 µg/ml), sucrose (100 µg/ml), mannan (1–100 µg/ml), or α-L-fucose (1–100 µg/ml). Cells were then washed, lysed and the binding of clusterin was revealed by ELISA. Results are expressed as the mean ± SD of 3 experiments (**p < .01, *p < .05). d. Macrophages were incubated for 15 min at 37°C with fucosylated clusterin (10 µg/ml) and clusterin endocytosis (green) was analyzed by confocal microscopy (n = 5, bars:10µm).

Article Snippet: Total clusterin was detected by ELISA using a conventional ELISA kit (Human Clusterin DuoSet ELISA, R&D Systems).

Techniques: Expressing, Derivative Assay, Incubation, Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

Journal: eLife

Article Title: Dynamics of compartment-specific proteomic landscapes of hepatotoxic and cholestatic models of liver fibrosis

doi: 10.7554/eLife.98023

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: rat mAb to K19 (Troma III; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); mouse mAbs to αSMA (DAKO, Glostrup, Denmark) and ECM1 (Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal antibodies to clusterin (R&D Systems, Minneapolis, MN, USA), rabbit polyclonal antibodies to fibronectin, collagen type I, collagen type IV, and monoclonal to integrin αV (all from Abcam, Cambridge, UK).

Techniques: Software, Staining