Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: The 51 genes within the functional annotation cluster were input into the STRING database. A network of how each gene interacts with others was produced. A sub-group of genes which interact closely, primarily through binding to each other, was highlighted (inset image). The genes of interest were CD247; LCK: Lymphocyte-specific protein tyrosine kinase; LAT: Linker of activated T cells; SYK: spleen tyrosine kinase; VAV2: VAV2 guanine nucleotide exchange factor; VAV3: VAV3 guanine nucleotide exchange factor.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Functional Assay, Produced, Binding Assay

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Gene expression levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by both human U133 plus 2 affymetrix gene assay and by quantitative reverse transcription polymerase chain reaction (PCR). Fold change from blood to lung is charted. Statistically significant differences between blood and pulmonary levels measured by PCR are indicated by *p<0.05, **p<0.001. Statistically significant differences between blood and pulmonary levels measured by microarray are indicated by † Q<0.01.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Gene Expression, Gene Assay, Reverse Transcription, Polymerase Chain Reaction, Microarray

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Pulmonary CD8 cells were isolated from chronic obstructive pulmonary disease (COPD, grey n = 6) and smokers with normal lung function (S, black, n = 6). Transcript levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by quantitative real time polymerase chain reaction. Statistically significant differences between COPD and S are represented by *p<0.05.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Expression Analysis of All Protease Genes Reveals Cathepsin K to Be Overexpressed in Glioblastoma

doi: 10.1371/journal.pone.0111819

Figure Lengend Snippet: (A) Upregulated expression of seven genes ( TFRC , CTSK , GFPT2 , ERAP2 , GPR56 , CD74 , PI3 ) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the CTSK gene using GBM tissues and cell lines with reference genes TBP and HPRT1 in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.

Article Snippet: Gene expression (RT-qPCR) analyses were performed using TaqMan Gene Expression Assays (Applied Biosystems): Hs00951083_m1 (for TFRC ), Hs00166156_m1 (for CTSK ), Hs01049561_m1 (for GFPT2 ), Hs01073631_m1 (for ERAP2 ), Hs00173754_m1 (for GPR56 ), Hs00269961_m1 (for CD74 ), Hs00160066_m1 (for PI3 ), and human GAPD ( GAPDH ) as a reference (No.: 4310884E) and ABI Prism 7900 Sequence Detection System (Applied Biosystems).

Techniques: Expressing, Microarray, Comparison, Quantitative RT-PCR, Standard Deviation

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: Primers and probes for TaqMan qRT-PCR

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Sequencing, Amplification

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: mRNA expression of PDGF ligands and receptors . mRNA expression of PDGF ligands and receptors calculated relative to mRNA expression levels of the endogenous control ( ACTB ) in A) non-euplastic thyroid tissue (NT, green) and a collection of classical (PTC(diff), blue) and clinically aggressive PTCs (PTC(agg), red). All values were adjusted so that the median value of the NT group was equal to one. Bars indicate median value of each individual group. P-values (below) are from Mann-Whitney t-test between groups, as indicated. B) Relative mRNA expression of all PDGF ligands and receptors in each individual biopsy. PDGFA (red triangle), PDGFB (green triangle), PDGFC (yellow square), PDGFD (brown circle), PDGFRA (magenta diamond) and PDGFRB (blue triangle). All values were adjusted so that the median value of the NT group was equal to one.

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Expressing, Control, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Inverse correlation between PDGFC expression and lymphocyte infiltration in human papillary thyroid carcinomas

doi: 10.1186/1471-2407-9-425

Figure Lengend Snippet: Quantification of PDGF expression level by Taman qRT-PCR and microarray hybridization . Expression profiles of A) PDGFA , B) PDGFB and C) PDGFRA accessed by two different techniques, Taman qRT-PCR (blue square) and cDNA microarray hybridization (magenta square) along the specimens investigated. Line is drawn between points for illustration purposes only. The three different groups of thyroid biopsy specimens are listed below the graph; Non-euplastic thyroid specimen (NT), differentiated PTC specimen (PTC (diff)) and poorly differentiated PTC (PTC (agg)).

Article Snippet: Applied Biosystems Assay On Demand, Hs00234042_m1 , 80 , NM_033016.1.

Techniques: Expressing, Quantitative RT-PCR, Microarray, Hybridization

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1-Dependent and Independent Function in Estrogen-Regulated Development in Vivo. A, Schematic depicting experimental procedure for ovariectomy and estrogen pellet implantation (n = 16 female mice). Mice were implanted with an estrogen pellet or placebo pellet 14 days after ovariectomy. Tissues were harvested at day 21. B, The representative images of uterus from cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. Graph depicts uterus weights as a percentage of body weight in cyclin D1+/+ and cyclin D1−/− mice with or without estrogen treatment. C, Mouse mammary gland whole mounts stained with Carmine dye.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, Staining

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genome-Wide Profiling of Cyclin D1-Dependent Estrogen-Regulated Genes in Vivo. A, Venn diagram displays the number of genes that were differentially regulated by estrogen in cyclin D1+/+ mouse mammary glands compared with cyclin D1−/− mouse mammary glands. The directionality of change is depicted by up and down arrows. B, Cyclin D1-dependent estrogen-regulated genes were grouped by hierarchical clustering via complete linkage (Cluster 3.0) and visually depicted using Treeview (left). The up-regulated genes are in red and down-regulated genes are in green (P < .05). Chart to the right of heat map depicts Log2 fold change of E2-induced and -repressed genes comparing cyclin D1+/+ with cyclin D1−/− mouse mammary glands. C–E, DAVID analysis was used to classify the pathways differentially regulated by cyclin D1 in E2-treated mice. Pathways depicted represent member genes from microarray analysis of estrogen-treated cyclin D1+/+ mouse mammary glands vs cyclin D1−/− mouse mammary glands, growth factors (C), growth factor receptors (D), and peptidases (E). V, vehicle; Veh., vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Genome Wide, In Vivo, Microarray

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Genes Regulated by E2 in Vivo Are Bound by Cyclin D1 by ChIP-Seq. A, Comparison of genes occupied by cyclin D1 in ChIP-Seq to those regulated by E2 in cyclin D1+/+ mouse mammary gland and (B) those E2-responsive genes that are regulated in a cyclin D1-dependant manner. C, Location of and (D) representative tag density profiles for, cyclin D1-occupied genes that are regulated by E2. Vertical axis shows average peak height and horizontal axis depicts chromosomal location of cyclin D1-associated interval sequence. TSS, transcription start site.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: In Vivo, ChIP-sequencing, Comparison, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Induces AREG Gene Expression in a Cyclin D1-Dependent Manner. A and B, MCF7 cells were transfected with siRNAs targeting cyclin D1 and treated with vehicle or E2 (10−8 M) for 24 hours. CCND1 and AREG mRNA abundance was determined by quantitative RT-PCR. C, Western blot was performed to determine the cellular levels of AREG expression in cyclin D1 knockdown cells compared with scramble siRNA control. β-tubulin was included as loading control for protein abundance. D, The concentration of AREG in cell culture medium was measured by ELISA. Concentration of AREG in the conditioned media was normalized to total protein. Data are mean ± SEM. E, AREG promoter luciferase reporter plasmids were transfected into MCF7 cells with a cyclin D1 expression vector. Relative luciferase activity is shown as mean ± SEM normalized to β-galactosidase activity of a cotransfected vector and as (F) fold-induction by cyclin D1. G, Promoter sequence alignment of mouse and human amphiregulin promoter. Homologous nucleotides (:) and regions of discontinuity (−) are indicated. Predicted BRCA1 sites are highlighted for human (− strand), and mouse (2 sites on + strand) with predicted confidence values of 86%, 87%, and 94% respectively. Ctrl., control.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Knockdown, Control, Quantitative Proteomics, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Activity Assay, Sequencing

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: Cyclin D1 Is Recruited to a BRCA1 Binding Site. A, ChIP assay in MCF7 cells treated with E2 (10 nM) for ERα at the AREG gene promoter. B. The pS2 gene was included as a positive control. C, Western blot shows FLAG-cyclin D1 expression in transduced MCF7 cells. D, ChIP assay to determine cyclin D1 occupancy at the AREG gene promoter. Using either MCF7 cells transduced with FLAG-cyclin D1 or (E) MCF7 cells ChIP analysis with antibodies directed to endogenous cyclin D1, BRCA1 or ERα (E2 10 nM 24 hours). F and G, GST pulldown was performed to determine the minimal region of BRCA1 required for cyclin D1 binding. H and I, GST-cyclin D1 or mutants were incubated with in vitro translated BRCA1. The N terminus (1–100 amino acids) of cyclin D1 was required for BRCA1 binding. IB, immunoblot; IP, immunoprecipitation; IVT, in vitro translation.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Binding Assay, Positive Control, Western Blot, Expressing, Transduction, Incubation, In Vitro, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: E2 Induces Cyclin D1 Distribution within a LMW Complex with AIB1. A,. Western blot analysis of Superose 6 chromatography from asynchronously cycling MCF7 cell lysates using antibodies as indicated to the left of the figure. The molecular weight of the fractions is indicated at the bottom of the figure. Cells were treated with (+) E2 (10−8 M). The coeluting fractions > 4 mDa (HMC) and 670 kDa (LMC) are indicated by the boxes. B, Western blot analysis of MCF7 cell extracts after Superose 6 chromatographic fractionation. The antibodies are as indicated. Extracts were treated with E2 (10−8M) for 30 minutes. Molecular weight markers are shown below panel B, indicating the HMC (4 MDa) or LMC (670 kDa). C, Hormone-deprived MCF7 cells infected with shCCND1 or shControl were treated for 1 hour with 10 nM E2 or vehicle control; ERα was immunoprecipitated followed by Western blotting for AIB1, cyclin D1, and ERα. IP, immunoprecipitation; V, vehicle.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Chromatography, Molecular Weight, Fractionation, Infection, Control, Immunoprecipitation

Journal: Molecular Endocrinology

Article Title: Cyclin D1 Determines Estrogen Signaling in the Mammary Gland In Vivo

doi: 10.1210/me.2013-1065

Figure Lengend Snippet: ERα Recruitment to the LMW Complex Requires Cyclin D1. A, Western blot analysis of superose 6 chromatographic fractions from female cyclin D1−/− or cyclin D1+/+ mice cell lysates (liver) using antibodies as indicated to the left of the figure. The molecular weight markers are shown below. B, The relative abundance of ERα in the HMC or LMC (670 kDa) is shown graphically indicating increased ERα in the HMC in cyclin D1−/− mice. C, Schematic presentation of cyclin D1 regulation of ERα activation proposes a model in which cyclin D1 participates in ERα signaling by binding to BRCA1 and in the presence of E2 facilitates an LMC that includes ERα and AIB1. Cyclin D1 binding to BRCA1 antagonized BRCA1 action, including BRCA1 repression of Areg expression.

Article Snippet: The antibodies used in Western blot analysis were to cyclin D1 (DCS-6), pRb (C-15), ERα (H-184), cyclin E (HE-12), CDK (C-22), pS2 (C-20) (Santa Cruz Biotechnology, Santa Cruz, California) and AREG (AF262; R&D Systems, Inc, Minneapolis, Minnesota).

Techniques: Western Blot, Molecular Weight, Activation Assay, Binding Assay, Expressing