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: Cell death & disease

Article Title: Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity.

doi: 10.1038/s41419-019-1857-x

Figure Lengend Snippet: Fig. 2 Inhibition of macroautophagy induction increased clusterin expression in MG132-treated cardiomyocytes. a, b Representative western blots and quantification of LC3 proteins levels, LC3II/LC3I ratio (a) and ubiquitinated proteins levels (b) in NCM treated with 3-methyladenine (3-MA, 10 mmol/L, 19 h) (n = 9) and/or MG132 (10 µmol/L, 18 h) (n = 9 and 13, respectively) compared to control cells (n = 13). c Analysis of clusterin (CLU) expression and secretion in NCM after the different treatments. Quantification by RT-qPCR, of CLU mRNA levels (upper panel) in the treated NCM (n = 8–9/group) compared to control cells (n = 8). Representative western blots and quantification of intracellular levels of precursor (p-CLU) and mature (m-CLU) forms of clusterin (n = 9 and 13/group, respectively) and its secreted form (s-CLU) (n = 8–9/group) in the treated NCM compared to control cells. For qPCR analysis, HPRT was used to normalize CLU expression and the data are expressed in arbitary units (A.U.). For western blot analysis, sarcomeric actin (S-actin) was used to normalize intracellular proteins levels and total mRNA levels were used to normalize secreted CLU levels. Data are expressed as individual and mean fold change in proteins levels relative to control cells. Statistical significance was determined by Wilcoxon–Mann Whitney test. *P < 0.05, ***P < 0.001 vs. control cells and #P < 0.05, ***P < 0.001 vs. MG132-treated cells

Article Snippet: To overexpress CLU, NCM and H9c2 cells were transfected with pCMV6 vector as a control or pCMV6Clu vector (OriGene, RN214684) using Lipofectamine 2000 (Invitrogen) with a ratio 1:2 (DNA/Lipo) according to the manufacturer’s instructions.

Techniques: Inhibition, Expressing, Western Blot, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cell death & disease

Article Title: Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity.

doi: 10.1038/s41419-019-1857-x

Figure Lengend Snippet: Fig. 3 Clusterin is degraded by proteasome and lysosome in cardiomyocytes. a Representative western blots and quantification of ubiquitinated proteins levels in NCM treated with cycloheximide (CHX, inhibitor of novo protein synthesis, 100 μg/mL) in presence or absence of MG132 (10 µmol/L) for 4 h compared to control cells (n = 6/group). b Representative western blots of LC3 and quantification of LC3II proteins levels in CHX-treated NCM in presence or absence of BAF (50 nmol/L) for 4 h compared to control cells (n = 6/group). c–e Representative western blots of intracellular CLU proteins levels and quantification of CLU mature form (m-CLU) levels in CHX-treated NCM in presence or absence of MG132 (c) or BAF (e) for 4 h compared to control cells (n = 6/group). For all intracellular proteins levels, sarcomeric actin (S-actin) was used to normalization. d–f Representative western blots and quantification of secreted CLU proteins levels (s-CLU) in CHX-treated NCM in presence or absence of MG132 (d) or BAF (e) for 4 h compared to control cells (n = 4/group). The culture medium is collected from the same cells after 24 h serum-deprivation corresponding to the beginning of treatment (T0h) and after 4 h of treatment (T4h). The levels of s-CLU quantified at T0h are used as normalizer. Graph show individual and mean fold change in proteins levels relative to control cells. Statistical significance was determined by Wilcoxon–Mann Whitney test. *P < 0.05, **P < 0.01 vs. control cells and #P < 0.05, ##P < 0.01 vs. CHX-treated cells

Article Snippet: To overexpress CLU, NCM and H9c2 cells were transfected with pCMV6 vector as a control or pCMV6Clu vector (OriGene, RN214684) using Lipofectamine 2000 (Invitrogen) with a ratio 1:2 (DNA/Lipo) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: Cell death & disease

Article Title: Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity.

doi: 10.1038/s41419-019-1857-x

Figure Lengend Snippet: Fig. 4 Clusterin silencing and overexpression had no effect on apoptosis induced by MG132 in cardiomyocytes. a Representative control- and MG132-treated NCM (10 µmol/L, 18 h) observed by phase-contrast microscopy. b Representative western blots and quantification of Bcl2 and cleaved caspase-3 proteins levels in MG132-treated NCM (n = 5) compared to control cells (n = 6). c NCM were transfected with siRNA non-target (si- NT) or siRNA targeting clusterin (siClu-1 and siClu-2) for 48 h before MG132 treatment (10 µmol/L, 18 h). Representative western blots and quantification of Bcl2 and cleaved caspase-3 proteins levels in MG132-treated NCM after CLU silencing (n = 8 and 6 for siClu-1 and siClu-2, respectively) compared to siNT-transfected NCM (n = 8). Sarcomeric actin (S-actin) was used to normalize proteins levels and data are expressed as individual and mean fold change in proteins levels relative to siNT-transfected cells. d NCM were transfected by empty vector (pCMV6) or vector overexpressing the secreted isoform of clusterin (pCMV6 Clu) for 48 h before MG132 treatment. Representative western blots and quantification of Bcl2 and cleaved caspase-3 proteins levels in transfected cells in presence and absence of MG132 (n = 6/group). Sarcomeric actin (S-actin) was used to normalize proteins levels and data are expressed as individual and mean fold change in proteins levels relative to pCMV6-transfected cells. e TUNEL staining (scale bar: 100 µm) in pCMV6 and pCMV6 Clu- transfected cells in presence or absence of MG132 and quantification of the percentage of TUNEL-positive cells in these 4 conditions normalized by the data obtained in pCMV6 group. (n = 9/group). Statistical significance was determined by Wilcoxon–Mann Whitney test. *P < 0.05, **P < 0.01 vs. nonstimulated condition

Article Snippet: To overexpress CLU, NCM and H9c2 cells were transfected with pCMV6 vector as a control or pCMV6Clu vector (OriGene, RN214684) using Lipofectamine 2000 (Invitrogen) with a ratio 1:2 (DNA/Lipo) according to the manufacturer’s instructions.

Techniques: Over Expression, Control, Microscopy, Western Blot, Transfection, Plasmid Preparation, TUNEL Assay, Staining, MANN-WHITNEY

Journal: Cell death & disease

Article Title: Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity.

doi: 10.1038/s41419-019-1857-x

Figure Lengend Snippet: Fig. 5 Increased clusterin expression in hypertrophied cardiomyocytes is associated with proteasome and macroautophagy inhibition. a Representative western blots and quantification of ubiquitinated proteins levels in NCM treated by isoproterenol (ISO, 10 µmol/L, 72 h) compared to control cells (n = 10/group). b Representative western blots of intracellular CLU proteins levels (precursor and mature forms) and its secreted form (top left panel) in NCM treated or not with ISO. Correlation of CLU precursor (top right panel), mature (bottom left panel) and secreted forms (bottom right panel) with ubiquitinated proteins levels in these cells. c Representative western blots and quantification of LC3 proteins levels and LC3II/LC3I ratio in ISO-treated NCM compared to control cells (n = 11/group). d Correlation of CLU precursor (left panel), mature (middle panel), and secreted form (right panel) with LC3II/LC3I ratio in NCM treated or not with ISO. For western blot analysis, sarcomeric actin (S-actin) was used to normalize proteins levels and data are expressed as individual and mean fold change in proteins levels relative to control cells. Statistical significance was determined by Wilcoxon–Mann Whitney or Spearman correlation test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. nonstimulated condition or indicated values

Article Snippet: To overexpress CLU, NCM and H9c2 cells were transfected with pCMV6 vector as a control or pCMV6Clu vector (OriGene, RN214684) using Lipofectamine 2000 (Invitrogen) with a ratio 1:2 (DNA/Lipo) according to the manufacturer’s instructions.

Techniques: Expressing, Inhibition, Western Blot, Control, MANN-WHITNEY

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: PLoS ONE

Article Title: Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: p15 and clusterin expression in human pituitary adenomas.

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

Techniques: Expressing

Journal: PLoS ONE

Article Title: Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: 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 αGSU in respective human pituitary adenoma specimens (clusterin green, respective hormones red).

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (sh Clu ) 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: A ) In vivo BrdU incorporation. Mice were injected with BrdU (50 µg/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 αGSU. PTTG pituitary glands, and in αGSU. PTTG pituitary adenomas; F ) Pituitary SA-β-galactosidase enzymatic activity (blue) in WT and in pre-tumorous αGSU. 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.

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (sh Clu ) 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: A ) C/EBPβ is up-regulated in the αGSU. PTTG pituitary. Confocal image showing C/EBPβ co-localization with αGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous αGSU. PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPβ–red, intranuclear); B ) Western blot analysis of C/EBPβ and δ isoforms induced in LβT2 cells stably transfected with m Pttg ; C ) Effects of C/EBPs on the clusterin promoter in LβT2 and αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and 800 ng murine pCDNA3-C/EBPα, β or δ. The ratio of luciferase to co-trasfected β-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 αT3 cells 24 hours after transfection with pCDNA3-C/EBPβ or E ) pCDNA3-C/EBPδ; F ) Western blot analysis of clusterin expression in LβT2 mPttg cells 48 hours after simultaneous transfection with siC/EBPβ and siC/EBPδ (3 nM each). Two different combinations of siRNAs were used.

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (sh Clu ) 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: A ) Effects of FOXL2 on the clusterin promoter in αT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-m Clu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co-trasfected β-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 αT3 cells 24 hours after transfection with pcDNA3-His-mFoxl2; C ) ChiP assay was performed in nuclear fractions derived from αT3 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.

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (sh Clu ) 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: FOXL2 directly activates the clusterin promoter, while Pttg overexpression results in proliferation, DNA damage and stimulation of C/EBPβ and δ; C/EBPs activate the clusterin promoter. High levels of secretory clusterin trigger expression of Cdk inhibitors p15, p16 and p27, and C/EBPβ 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.

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

Techniques: Over Expression, Expressing