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: Human reproduction (Oxford, England)

Article Title: CDC42 deficiency leads to endometrial stromal cell senescence in recurrent implantation failure.

doi: 10.1093/humrep/deae246

Figure Lengend Snippet: Figure 1. Cellular senescence in the stroma of recurrent implantation (RIF) failure endometrium. (A) Gene ontology (GO) biological process enrichment of the differentially expressed genes (DEGs) between fertile control (CTR) patients and patients with RIF in SRP224538. (B) FPKM level of TP53 in CTR patients and RIF patients in SRP224538. (C) FPKM level of IL6 in CTR patients and RIF patients in SRP224538. (D–F) Levels of IL6, CLU, and sST2 secretion in primary endometrial stromal cell supernatant from CTR and RIF patients before and after 72 h of 8Br-cAMPþMPA treatment. (G and H) Representative images of immunohistochemical (IHC) staining of P16 in mid-secretory endometrium of CTR patients (n ¼ 22) versus RIF patients (n ¼ 22). Black arrows indicate typical P16þ cells. Quantitative analysis by number of P16þ cells per 200× field for P16 staining. (I and J) Representative images of IHC staining of P21 in mid-secretory endometrium of CTR patients (n ¼ 22) versus RIF patients (n ¼ 22). Black arrows indicate typical P21þ

Article Snippet: Commercially available ELISA kits were utilized to measure the level of secreted protein (PRL: Elabscience, No. E-EL-H0141; IGFBP1: Elabscience, No. E-EL-H0442; IL6: Elabscience, No. E-EL-H6156; IL8: MULTI SCIENCES, No. EK10896; CLU: Elabscience, No. E-EL-H0038; sST2: Elabscience, No. E-EL-H6082) in collected supernatants according to the manufacturer’s instructions.

Techniques: Control, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Human reproduction (Oxford, England)

Article Title: CDC42 deficiency leads to endometrial stromal cell senescence in recurrent implantation failure.

doi: 10.1093/humrep/deae246

Figure Lengend Snippet: Figure 2. CDC42 knockdown accelerates senescence of differentiated endometrial stromal cells (EnSCs). (A–C) Expression of CDC42, CDKN2A, and CDKN1A mRNA levels in primary EnSCs after 72 h of adenovirus treatment. (D) Expression of CDC42, P21, P53, and p-γ-H2AX protein levels in primary EnSCs after 72 h of adenovirus treatment. (E) Expressions of collagen I, collagen III, collagen IV, and MMP2 protein levels in primary EnSC after 72 h of adenovirus treatment. (F–H) Expressions of CDC42, CDKN2A, and CDKN1A mRNA levels in primary EnSC transfected with Ad-GFP or Ad-shCDC42-GFP following with 72 h of 8Br-cAMPþMPA treatment. (I–K) Level of IL6, CLU, and sST2 secretion in primary EnSCs transfected with Ad-GFP or Ad-shCDC42- GFP following with 72 h of 8Br-cAMPþMPA treatment. (L) Expression of CDC42, P21, and P53 protein levels in primary EnSCs transfected with Ad-GFP or Ad-shCDC42-GFP following with 72 h of 8Br-cAMPþMPA treatment. (M and N) Representative images of P16 and P21 immunofluorescence staining in primary EnSCs transfected with Ad-GFP or Ad-shCDC42-GFP following with 72 h of 8Br-cAMPþMPA treatment. (O and P) SA-β-gal staining of primary EnSCs after 48 h of Ad-GFP or Ad-shCDC42-GFP adenovirus treatment and then different days of 8Br-cAMPþMPA treatment. Quantitative analysis of integrated optical density for SA-β-gal staining, with value of the Ad-GFP group at 0 h set as 1. Mean ± SEM. P < 0.05, P < 0.01, P<0.001. ANOVA with Tukey’s multiple comparisons test. Two-way ANOVA with the Bonferroni multiple comparisons test in (P).

Article Snippet: Commercially available ELISA kits were utilized to measure the level of secreted protein (PRL: Elabscience, No. E-EL-H0141; IGFBP1: Elabscience, No. E-EL-H0442; IL6: Elabscience, No. E-EL-H6156; IL8: MULTI SCIENCES, No. EK10896; CLU: Elabscience, No. E-EL-H0038; sST2: Elabscience, No. E-EL-H6082) in collected supernatants according to the manufacturer’s instructions.

Techniques: Knockdown, Expressing, Transfection, Immunofluorescence, Staining

Journal:

Article Title: Temporal Accrual of Complement Proteins in Amyloid Plaques in Down's Syndrome with Alzheimer's Disease

doi:

Figure Lengend Snippet: Conditions, Sources, and Manufacturers of Antibodies Used

Article Snippet: Apo-J , Apo-J , Goat anti-human , 1:200 , Microwave/proteinase K , Rockland, Gilbertsville, PA.

Techniques: Labeling

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer

doi: 10.1007/s00018-017-2631-9

Figure Lengend Snippet: IPA networks of direct relationships. IPA networks of 14 proteins differentially expressed a between healthy controls vs. CRC patients (early- and late-stage) with a score of 40 and b between healthy controls vs. early-stage CRCs with a score of 41. Red marked proteins are lower expressed in CRC platelet samples compared to healthy controls, whereas green highlighted proteins are higher expressed in cancer samples. Out of these networks, clusterin (gene symbol: CLU) and glutathione synthetase (gene symbol: GSS) were selected for subsequent validation

Article Snippet: The membrane was blocked at room temperature for 1 h with 2% Amersham ECL Prime Blocking Agent (GE Healthcare), dissolved in 1× TBS with 0.1% Tween-20 (pH 7.6, Cell Signaling, USA) and incubated with primary antibodies against clusterin (rabbit monoclonal antibody, clone EPR2911, TA307501, OriGene), cofilin-1 (mouse monoclonal antibody, clone E-8, sc-376476, Santa Cruz Biotechnology, Inc., USA) and glutathione synthetase (rabbit polyclonal antibody, clone H-300, sc-28966, Santa Cruz Biotechnology) in 2% blocking buffer at 4 °C overnight.

Techniques: Biomarker Discovery

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer

doi: 10.1007/s00018-017-2631-9

Figure Lengend Snippet: Western blot validation of platelet target proteins clusterin, GSH-S, and cofilin-1. Specific antibody-targeted protein bands were detected by Cy3-labeled secondary antibody. Cy5 total protein signals within each lane were used for normalization (Cy3/Cy5 ratio). Based on an internal standard, adjusted relative protein-level calculation was performed. Western blot validation of selected target proteins a clusterin, c GSH-S, and e cofilin-1 confirmed significant different level characteristics obtained by 2D-DIGE between distinct groups (p < 0.05). Based on ROC curve calculation, cut-offs for clusterin, GSH-S, and cofilin-1 were selected grouping values of protein levels into healthy control or early-stage CRC patient. Red lines represent median values. Maximal sensitivity is indicated by dashed lines and best specificity by dotted lines. a sensitivity; b specificity

Article Snippet: The membrane was blocked at room temperature for 1 h with 2% Amersham ECL Prime Blocking Agent (GE Healthcare), dissolved in 1× TBS with 0.1% Tween-20 (pH 7.6, Cell Signaling, USA) and incubated with primary antibodies against clusterin (rabbit monoclonal antibody, clone EPR2911, TA307501, OriGene), cofilin-1 (mouse monoclonal antibody, clone E-8, sc-376476, Santa Cruz Biotechnology, Inc., USA) and glutathione synthetase (rabbit polyclonal antibody, clone H-300, sc-28966, Santa Cruz Biotechnology) in 2% blocking buffer at 4 °C overnight.

Techniques: Western Blot, Biomarker Discovery, Labeling, Control

Journal: Cancer science

Article Title: Immunophenotypic features of metastatic lymph node tumors to predict recurrence in N2 lung squamous cell carcinoma.

doi: 10.1111/cas.12434

Figure Lengend Snippet: Fig. 3. Kaplan–Meier recurrence-free survival (RFS) curve for patients with resected pathological N2 squamous cell carcinoma of the lung according to immunohistochemical staining. (a) Kaplan–Meier RFS curve according to clusterin expression of cancer cells in metastatic lymph node tumors. (b) Kaplan–Meier RFS curve according to ZEB2 expression of cancer cells in metastatic lymph node tumors. (c) Kaplan–Meier RFS curve according to podoplanin expression of cancer-associated fibroblasts in metastatic lymph node tumors.

Article Snippet: Caveolin (clone D46G3; Cell Signaling, Danvers, MA, USA),(8,9) clusterin (clone 1A11; Acris Antibodies, Herford, Germany),(10,11) E-cadherin (clone 36; BD Biosciences, San Jose, CA, USA),(12,13) and ZEB2 (Novus Biologicals, Littleton, CO, USA)(14,15) were used as EMT-related markers.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Scientific reports

Article Title: Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

doi: 10.1038/srep29521

Figure Lengend Snippet: Figure 4. Clusterin mediates selective survival of Ly6C+ MDSCs. (A) Western blot analysis of secretory/ cytoplasmic clusterin (sCLU) in Ly6G+ G-MDSC and Ly6C+ M-MDSC isolated from tumor bearers. Both the 60 kD and the 39/41 kD forms can be detected in Ly6C+ but not Ly6G+ cells. Actin was used as loading control. (B,C) Ly6C+ M-MDSC or RAW264.7 macrophages were transfected with antisense-scramble or antisense-CLU for 24 h, and then tranfection efficiency was checked by analysis of sCLU expression by Q-PCR (B) and western blot (C) analysis. (D) Analysis of apoptosis by AnnexinV+ staining indicated that knockdown of sCLU by siRNA- CLU induced apoptosis in both RAW264.7 cells and M-MDSCs treated with 20 uM curcumin or 10 ng/ml DTX. Bar graphs show mean ± SE of three independent experiments. *P < 0.05.

Article Snippet: Clusterin siRNA and Scrambled negative control were purchased from OriGene (Rockville, MD).

Techniques: Western Blot, Isolation, Control, Transfection, Expressing, Staining, Knockdown

Journal: Scientific reports

Article Title: Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

doi: 10.1038/srep29521

Figure Lengend Snippet: Figure 5. Clusterin prevents apoptosis by inhibiting Bax translocation to mitochondria (A) sCLU immunoprecipitated from Ly6C+ MDSCs treated or untreated 12 h with 20 uM curcumin (CUR) or 10 ng/ml DTX were subjected to western blot analysis with 6A7 anti-active Bax antibody. sCLU did not bind active Bax in control DMSO-treated Ly6C+ cells but did so after curcumin or DTX treatment. (B) RAW264.7 macrophages were transfected with antisense-scramble or CLU for 24 h and then treated with DMSO, 20 μM curcumin or 10 ng/ml DTX for 2 h. Cells were incubated with 125 nM MitoTracker Red CMXRos for 30 min at 37 °C (red fluorescence), then stained with anti-6A7 followed by Alexa Fluor 488-conjugated antibody (green fluorescence). Cells were examined by confocal microscopy (magnification, ×630). Active Bax shows little translocation to mitochondria in scrambled control but almost complete colocalization in anti-sense CLU-transfected macrophages upon curcumin or DTX treatment. One representative set of images of three independent experiments is shown.

Article Snippet: Clusterin siRNA and Scrambled negative control were purchased from OriGene (Rockville, MD).

Techniques: Translocation Assay, Immunoprecipitation, Western Blot, Control, Transfection, Incubation, Fluorescence, Staining, Confocal Microscopy

Journal: Scientific reports

Article Title: Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

doi: 10.1038/srep29521

Figure Lengend Snippet: Figure 6. Clusterin is expressed in mature macrophages and not in immature myeloid cells in human breast tumor tissues from patients with invasive ductal carcinoma. Twenty formalin-fixed paraffin- embedded human normal breast tissues (A) and breast cancer samples (B) were stained for sCLU and the numbers of macrophages (red circle) and immature myeloid cells (black circle) were visually assessed by morphology. The intensity of sCLU staining was scored from 0 to 3, with 0 being the lowest, which is shown next to each circle. Note that the breast cancer cells are all sCLU positive, as has been reported by others. (C) The twenty samples from normal and breast cancer were then analyzed for the average distribution of mature and immature myeloid cells expressing sCLU and the total number of mature macrophages or immature macrophage cells per tissue sample is shown. (D) Intensity of sCLU staining for each macrophage or immature myeloid cells per tissue sample. P values (***P < 0.001) were generated by Student’s t test.

Article Snippet: Clusterin siRNA and Scrambled negative control were purchased from OriGene (Rockville, MD).

Techniques: Formalin-fixed Paraffin-Embedded, Staining, Expressing, Generated