Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-?-mediated Epithelial-Mesenchymal Transition *

doi: 10.1074/mcp.M110.001131

Figure Lengend Snippet: Selected proteins differentially expressed during Ras/TGF-β-induced EMT

Article Snippet: Following incubation, medium was replaced with serum-free DMEM containing 20 ng/ml recombinant mouse Wnt-3a (R&D Systems), 20 or 40 ng/ml recombinant mouse Wnt-5a (R&D Systems), or 30% conditioned medium from 21D1 cells.

Techniques: Microarray, Transduction, Transformation Assay

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-?-mediated Epithelial-Mesenchymal Transition *

doi: 10.1074/mcp.M110.001131

Figure Lengend Snippet: siRNA silencing of Wnt-5a attenuates 21D1 cell migration and invasion. A, transcript expression of Wnt-5a was not detected in MDCK cells by semiquantitative RT-PCR; however, transformation with H-Ras and stimulation with TGF-β induces Wnt-5a expression. B, expression of Wnt-5a in 21D1 cells is attenuated by RNA interference using siRNA duplexes targeting Wnt-5a. The addition of scrambled negative control (NC) siRNA did not affect Wnt-5a expression. Expression of Wnt-5a was reduced in 21D1 cells using transient siRNA transfection, and cell migration was assessed using the wound healing (C) and Transwell migration (D) assays. Both assays show that cell migration is significantly impeded when expression of Wnt-5a is reduced. However, cell migration is restored and even elevated with the addition of rm Wnt-5a (n = 2). E, the ability of 21D1 cells to invade through the matrix is decreased when expression of Wnt-5a is reduced using siRNA duplexes, although this reduction is not statistically significant (**). The invasive nature of 21D1 cells is rescued with the addition of recombinant Wnt-5a (n = 2). Error bars represent mean ± S.D. Significance is determined by a p value ≤0.05 using the Student's t test.

Article Snippet: Following incubation, medium was replaced with serum-free DMEM containing 20 ng/ml recombinant mouse Wnt-3a (R&D Systems), 20 or 40 ng/ml recombinant mouse Wnt-5a (R&D Systems), or 30% conditioned medium from 21D1 cells.

Techniques: Migration, Expressing, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Negative Control, Transfection, Recombinant

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-?-mediated Epithelial-Mesenchymal Transition *

doi: 10.1074/mcp.M110.001131

Figure Lengend Snippet: Transcription factors up-regulated during EMT that are predicted to bind promoter of Wnt-5a HMG, high mobility group.

Article Snippet: Following incubation, medium was replaced with serum-free DMEM containing 20 ng/ml recombinant mouse Wnt-3a (R&D Systems), 20 or 40 ng/ml recombinant mouse Wnt-5a (R&D Systems), or 30% conditioned medium from 21D1 cells.

Techniques: Microarray

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomics Profiling of Madin-Darby Canine Kidney Plasma Membranes Reveals Wnt-5a Involvement during Oncogenic H-Ras/TGF-?-mediated Epithelial-Mesenchymal Transition *

doi: 10.1074/mcp.M110.001131

Figure Lengend Snippet: Wnt-5a expression during EMT represses canonical Wnt signaling. A, canonical Wnt signaling activity was examined using the T-cell factor-luciferase reporter assay under different culture conditions. 21D1 cells cultured in 30% conditioned medium (CM) showed no significant change (**) in canonical Wnt signaling activity. Addition of rm Wnt-3a stimulates canonical Wnt signaling; however, this activity is significantly (*) repressed following the addition of 20 ng/ml and to a greater extent by 40 ng/ml recombinant Wnt-5a. In a similar fashion, addition of 30% conditioned medium from 21D1 cells significantly attenuates canonical Wnt signaling (n = 2). Error bars represent mean ± S.D. Significance is determined by a p value ≤0.05 using the Student's t test. B, the non-canonical Wnt signaling PCP pathway may be activated during Ras/TGF-β-induced EMT. Several upstream components, core components, downstream effectors, and PCP modulators were revealed to be up-regulated in our integrated proteomics and transcriptomics EMT studies. Cthrc1, collagen triple helix repeat-containing protein 1; Fzd6, frizzled 6; Ptk7, tyrosine-protein kinase 7; DVL, dishevelled; ND, not detected; NC, no change. Pathway interactions are based on information obtained from the Kyoto Encyclopedia of Genes and Genomes database (http://www.genome.jp/kegg/pathway.html).

Article Snippet: Following incubation, medium was replaced with serum-free DMEM containing 20 ng/ml recombinant mouse Wnt-3a (R&D Systems), 20 or 40 ng/ml recombinant mouse Wnt-5a (R&D Systems), or 30% conditioned medium from 21D1 cells.

Techniques: Expressing, Activity Assay, Luciferase, Reporter Assay, Cell Culture, Recombinant

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Localization of clusterin in normal human lung. Clusterin was detected immunohistochemically by staining formalin-fixed, paraffin embedded 3 μm sections of human control lung tissue. Representative images of clusterin (clu, A-C, brown/red, nuclei - blue) and elastic fibers (( D ), grey/black) in tissue obtained from control lung (n = 3). Clusterin localizes to fibroblast-like cells ( A ), to small areas of bronchial epithelial cells ( B ) and to elastic fibers in blood vessels and alveolar walls ( C , D ) serial sections). Clusterin was not detectable in macrophages, alveolar epithelial cells ( A ) or endothelial cells ( C ). Different cell populations/structures are indicated by arrows: f - fibroblast-like cell, m - macrophage, e – alveolar epithelial cell, be - bronchial epithelial cell, en - endothelial cell, smc – smooth muscle cell, ef - elastic fibers. Scale bar represents 25 µm.

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Staining, Formalin-fixed Paraffin-Embedded, Control

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Localization of clusterin in IPF lung. Immunohistochemical staining for clusterin was performed on formalin-fixed, paraffin embedded 3 μm sections of IPF lung tissue (n = 3). Clusterin staining (clu, A, C-G, I brown/red, nuclei - blue) and staining for elastin (H, J, EvG, grey/black) in representative tissue sections. Clusterin is undetectable in αSMA positive myofibroblasts ( A,B ), forming and in cells overlying fibroblastic foci ( A,C ), compared to strong staining of fibroblast-like cells in morphologically normal non-fibrotic areas ( D ). Clusterin was observed sporadically in bronchial epithelial cells but more frequently than in controls ( E ). Similar to control lung, clusterin colocalized with elastin ( G,H ) and was undetectable in macrophages, smooth muscle and endothelial cells ( F,G ). Clusterin also colocalized with amorphous elastin aggregates in dense fibrotic regions ( I,J ). Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, be - bronchial epithelial cell, en – endothelial cell, smc – smooth muscle cell, ef- elastic fibers. Scale bar represents 25 µm ( A–J ).

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Control

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Clusterin gene expression and protein levels are decreased in fibrotic compared with control lung fibroblasts. Fibroblasts isolated from human control and fibrotic lung were grown in monolayer culture and clusterin mRNA and protein levels were detected via microarray, proteome profiler and immunofluoresecence analysis. ( A ) Microarray analysis of mRNA shows decreased clusterin gene expression in fibroblasts derived from fibrotic lungs (open circles; n = 5 IPF and 7 SSc) compared with controls (closed circles; n = 6). Proteome profiler array analysis ( B , clusterin - CLU) and immunofluorescence staining of control and fibrotic lung fibroblasts ( C,D ) confirms low clusterin protein expression in fibrotic compared with control fibroblasts in vitr o. ( E ) Semi-quantitative analysis of clusterin staining ( C,D ), clusterin signal (pixel intensity) normalized to cell numbers per visual field (n = 6). Data is representative of three individual experiments. * P < 0.05, **** P < 0.0001; Scale bar in D represents 10 µm.

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Gene Expression, Control, Isolation, Microarray, Derivative Assay, Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: TGF-β 1 associates with areas of decreased clusterin expression in fibrotic lung and down-regulates fibroblast clusterin mRNA and protein expression in vitro . Serial sections prepared from IPF lung (n = 3) were stained immunohistochemically to localize TGF-β 1 staining (( A ), red/brown, nuclei blue) and clusterin (( B ), red/brown, nuclei blue) in fibroblastic foci. ( A,B ) Representative images of immunohistochemical staining suggest that TGF-β 1 localizes to ECM, fibroblasts and macrophages, whilst staining for clusterin is weak or undetectable. ( C ) In vitro analysis of clusterin mRNA levels in TGF-β 1 stimulated (40 pM) human lung fibroblasts was performed via qRT-PCR and shows a time-dependent down-regulation of clusterin expression that was maximal at 24–48 h (n = 3) in response to TGF-β 1 . Clusterin protein levels were also down-regulated in response to TGF-β 1 (40 pM) compared to control at 24 h and 48 h as demonstrated by western blotting at 24 h ( D ) and immunofluorescent staining at 48 h (E, red, nuclei - blue). ( F ) Semi-quantitative analysis of fluorescent signal of panel E: clusterin signal (pixel intensity) was normalized to cell numbers per visual field and compared to control (n = 6). Full-length western blots are presented in Supplementary Figure . Different cell populations/structures are indicated by arrows; f - fibroblast-like cell, m - macrophage, he - hyperplastic epithelial cell, ecm – extracellular matrix. * P < 0.05, ** P < 0.01; scale bar 100 µm (A) and 10 µm ( E ).

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Expressing, In Vitro, Staining, Immunohistochemical staining, Quantitative RT-PCR, Control, Western Blot

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on TGF-β 1 -induced myofibroblast differentiation and collagen deposition. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untransfected (black bars). αSMA mRNA was assessed via qRT-PCR ( A ) and clusterin and αSMA protein levels detected by western blotting ( B,C quantification). 48 h following TGF-β 1 stimulation (40 pM) αSMA mRNA and protein were increased. Basal and increased levels of αSMA mRNA and protein, however, did not vary between clusterin deficient, mock-transduced and control fibroblasts. Collagen mRNA ( D ) and deposition ( E , F quantification) assessed by qRT-PCR and immunofluorescence staining were significantly increased in response to TGF-β 1 . Although, basal and TGF-β 1 induced changes in collagen levels varied between clusterin deficient, mock and control fibroblasts, the overall fold-increase in collagen mRNA and deposition levels did not significantly change between clusterin deficient fibroblasts and control/mock fibroblasts. Data generated in A-F is representative of two individual experiments with fibroblasts derived from 2 donors. Full-length western blots are presented in Supplementary Figure . Scale bar in E represents 10 µm. ** P < 0.01, *** P < 0.001 compared with untreated controls respectively.

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Transduction, shRNA, Quantitative RT-PCR, Western Blot, Control, Immunofluorescence, Staining, Generated, Derivative Assay

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on lung fibroblast proliferation.Lung fibroblasts were transduced with clusterin shRNA (shCLU, open bars) and mock shRNA vectors (grey bars) or remained untreated (black bars). Proliferation in shRNA-mediated clusterin deficient fibroblasts ( A ) or fibrotic lung fibroblasts ( B ) compared with controls was assessed in response to the indicated stimuli for 48 h or 72 h for FBS by counting DAPI-positive nuclei in a high-throughput immunofluorescence assay. Cell numbers were normalized to cell counts of controls (0.4% FBS in DMEM) and expressed as percent change in proliferation relative to control (n = 6). Data representatives of two individual experiments with fibroblasts derived from 1 donor per group. Significances compared with controls are marked with (#) symbol and significances between controls vs. shCLU or non-fibrotic vs. fibrotic lung fibroblasts are indicated with (*). */# P < 0.05, **/## P < 0.01, ***/ ### P < 0.001, ****/ #### P < 0.0001.

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Transduction, shRNA, High Throughput Screening Assay, Immunofluorescence, Control, Derivative Assay

Journal: Scientific Reports

Article Title: Diverse functions of clusterin promote and protect against the development of pulmonary fibrosis

doi: 10.1038/s41598-018-20316-1

Figure Lengend Snippet: Effect of clusterin deficiency on apoptosis. Lung fibroblasts were transduced with clusterin shRNA (shCLU, open circles) and mock shRNA vectors (grey circles) or remained untransfected (black circles). Lung fibroblasts were seeded and treated with FasL (3 nM – 6 nM) and/or exogenous clusterin (CLU, 125 nM) for 19 h or remained untreated. Apoptotic cells (Annexin V+ and Annexin V+/ DAPI+ cells) were assessed via FACS analysis post staining of apoptotic cells with annexin V – Alexa647 and DAPI (mean ± SEM, n = 5). ( A ) shRNA-induced clusterin deficiency sensitized fibroblasts to basal and FasL-induced apoptosis, and this could be overcome by addition of exogenous clusterin ( B ). ( C ) Basal apoptosis in representative control (9.37 ± 0.63%) and fibrotic (11.6 ± 0.69%) lung fibroblast isolates was not significantly different. Fibrotic lung fibroblasts were more resistant to FasL-induced apoptosis compared with controls ( C ) and exogenous clusterin tends to reduce basal and FasL-induced apoptotic levels further ( D ). Data representative of at least two individual experiments with fibroblasts derived from 1 donor per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Separate wells of cells were incubated overnight with monoclonal mouse antibodies against collagen type I (Sigma, Aldrich, C2456 at 4.7 μg/ml) or mouse monoclonal against clusterin-α (Santa Cruz, sc-5289, at 2.0 μg/ml) and then washed three times with 0.05% (v/v) Tween in PBS (PBS-T).

Techniques: Transduction, shRNA, Staining, Control, Derivative Assay

Journal: Frontiers in Immunology

Article Title: lncRNA-HEIM Facilitated Liver Fibrosis by Up-Regulating TGF- β Expression in Long-Term Outcome of Chronic Hepatitis B

doi: 10.3389/fimmu.2021.666370

Figure Lengend Snippet: Identification of differentially expressed lncRNAs correlated with TGF- β signaling pathway in long-term CHB patients. (A) Hierarchical clustering analysis of 4,718 differentially expressed lncRNA transcripts between CHB with long-term antiviral treatment and ASC samples (fold change >2, up- or down-regulated). Columns represent each gene, and rows represent each sample. The relative expression levels of mRNAs are depicted into color scale ranging from green (down-regulated) to red (up-regulated). (B) Alluvial diagram of candidate differentially expressed lncRNAs with up-regulated mRNAs from enriched KEGG pathways. (C) Pairwise correlations between lncRNAs and mRNAs (upper), with a color gradient denoting Pearson correlation coefficients. Pairwise comparisons between lncRNAs and mRNAs (lower). Edge width corresponds to the Mantel’s r statistic, and edge color denotes the statistical significance. (D) Real-time PCR validation of lncRNA ENST00000519726 in ASC or CHB samples. ∗ p < 0.05.

Article Snippet: Each labeled cRNA was fragmented and hybridized on Human LncRNA Microarray V4.0 (Arraystar, containing 40,173 annotated lncRNAs or lncRNAs of high confidence, as well as an entire collection of 20,730 protein coding mRNAs).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: lncRNA-HEIM Facilitated Liver Fibrosis by Up-Regulating TGF- β Expression in Long-Term Outcome of Chronic Hepatitis B

doi: 10.3389/fimmu.2021.666370

Figure Lengend Snippet: lncRNA-HEIM ENST00000519726 was highly expressed in monocytes. (A) Relative expression levels of SMAD4 and TGF- β in CD14 + monocytes against those in CD14 − cells from CHB patients. (B) Relative expression levels of lncRNA ENST00000519726 in CD14 + monocytes against those in CD14 - cells from either healthy donor (HD) or CHB patients. (C) Relative expression levels of lncRNA-HEIM, SMAD4, and TGF- β in monocytes at 2, 8, 16, and 24 h upon HBV infection. Expression levels of lncRNA-HEIM, SMAD4, and TGF- β were normalized with negative control at each time point. Comparisons over groups were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. ∗ p < 0.05. Blue line is for lncRNA-HEIM, while green and red lines are for SMAD4 and TGF- β , respectively. (D) Relative expression levels of lncRNA-HEIM, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and U6 in cytoplasm or nuclear of monocytes. GADPDH was used as cytoplasmic control transcripts, and U6 as nuclear control. (E) Relative expression levels of lncRNA-HEIM transfected with negative control or siRNAs targeting lncRNA-HEIM (silncRNAs). **p < 0.01, ***p < 0.001.

Article Snippet: Each labeled cRNA was fragmented and hybridized on Human LncRNA Microarray V4.0 (Arraystar, containing 40,173 annotated lncRNAs or lncRNAs of high confidence, as well as an entire collection of 20,730 protein coding mRNAs).

Techniques: Expressing, Infection, Negative Control, Control, Transfection

Journal: Frontiers in Immunology

Article Title: lncRNA-HEIM Facilitated Liver Fibrosis by Up-Regulating TGF- β Expression in Long-Term Outcome of Chronic Hepatitis B

doi: 10.3389/fimmu.2021.666370

Figure Lengend Snippet: Over-expression of lncRNA-HEIM remarkably promoted the expression of TGF- β and SMAD4. (A) Relative expression of SMAD4 or TGF- β in monocytes transfected with siRNAs for lncRNA-HEIM or negative control (NC). ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001. (B) Western blotting assays of endogenous protein levels of TGF- β , SAMD4, phosphorylated SMAD2, 3, and total SMAD2, 3 in monocytes transfected with siRNAs for lncRNA-HEIM or NC. GAPDH protein levels were used as internal controls. Chemiluminescent densities were normalized with GAPDH, and fold changes were calculated based on NC. (C) ELISA based TGF- β levels in culture supernatant of monocytes transfected with siRNAs for lncRNA-HEIM or NC. ∗ p < 0.05. (D) Relative expression of lncRNA-HEIM in monocytes infected with lncRNA-HEIM lentivirus (LV-HEIM) or negative control (NC). (E) Relative expression of TGF- β or SMAD4 in monocytes stably over-expressing lncRNA-HEIM or NC. ∗ p < 0.05; ∗∗∗ p < 0.001. (F) ELISA based TGF- β levels in culture supernatant of monocytes with LV-HEIM or NC. ∗∗ p < 0.01. (G) Western blotting assays of endogenous protein levels of TGF- β , SAMD4 in monocytes with LV-HEIM or NC. Fold changes, same as above.

Article Snippet: Each labeled cRNA was fragmented and hybridized on Human LncRNA Microarray V4.0 (Arraystar, containing 40,173 annotated lncRNAs or lncRNAs of high confidence, as well as an entire collection of 20,730 protein coding mRNAs).

Techniques: Over Expression, Expressing, Transfection, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Stable Transfection

Journal: Frontiers in Immunology

Article Title: lncRNA-HEIM Facilitated Liver Fibrosis by Up-Regulating TGF- β Expression in Long-Term Outcome of Chronic Hepatitis B

doi: 10.3389/fimmu.2021.666370

Figure Lengend Snippet: lncRNA-HEIM dramatically facilitated the fibrosis of hepatic stellate cells. (A, B) Relative mRNA expression and western blotting assays of collagen I and α -smooth muscle actin ( α -SMA) in hepatic stellate cells (HSCs) co-cultured with monocytes transfected with siRNAs for lncRNA-HEIM or NC. Chemiluminescent densities in (B) were normalized with GAPDH, and fold changes were calculated based on NC. ∗ p < 0.05. (C, D) Relative mRNA expression and western blotting assays of collagen I and α -smooth muscle actin ( α -SMA) in HSCs co-cultured with LV-HEIM stable monocytes or NC. Fold changes in (D) , same as above. ∗∗ p < 0.01. (E) Confocal imaging of collagen-I (red), α -SMA (green), and DAPI (blue) in HSCs co-cultured with LV-HEIM stable monocytes or NC. Scale bar: 20 μm. Relative intensity of density normalized by DAPI was depicted on the right. ∗∗∗ p < 0.001.

Article Snippet: Each labeled cRNA was fragmented and hybridized on Human LncRNA Microarray V4.0 (Arraystar, containing 40,173 annotated lncRNAs or lncRNAs of high confidence, as well as an entire collection of 20,730 protein coding mRNAs).

Techniques: Expressing, Western Blot, Cell Culture, Transfection, Imaging

Journal: Oncotarget

Article Title: Ameloblastoma RNA profiling uncovers a distinct non-coding RNA signature

doi: 10.18632/oncotarget.13889

Figure Lengend Snippet: Total RNA-extracted (DNAse treated) from 35 ameloblastoma samples and 21 non-ameloblastoma oral tissues were tested in qPCR using site-specific primers for each ncRNA candidate showing significantly increased relative expression in microarray or high cancer relevance. Dot plots show the individual ncRNA fold expression level of each tumour sample relative to the expression level of control group, including the group mean values, standard deviations and p -values. To normalise the ncRNA expression level, internal controls, GAPDH and RNU48 were used. Unpaired 2-tailed t -tests, was used for statistical comparison between the groups, statistical significance was defined as p -value < 0.05.

Article Snippet: Among the 245 000 human protein-coding transcripts and the 40 000 known ncRNA probes derived from RefSeq, Ensemble, IncRNAdb and Broad Institute, the microarray assay captured 85 significantly up-regulated ( p -value < 0.05 and FC > 2.0) and 36 down-regulated ( p -value < 0.05 and FC <−2.0) protein-coding transcripts in the ameloblastoma samples ( ).

Techniques: Expressing, Microarray, Control, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: O-antigen and Virulence Profiling of Shiga Toxin-Producing Escherichia coli by a Rapid and Cost–Effective DNA Microarray Colorimetric Method

doi: 10.3389/fcimb.2012.00061

Figure Lengend Snippet: Schematic diagram of ampliPHOX colorimetric method with DNA microarrays . First, DNA microarrays are hybridized with DNA target labeled with biotin (red circles). Second, the microarray is labeled with a photoinitiator (letter P) that is conjugated to streptavidin (blue polygons). Third, a short light-initiated polymerization reaction results in a colorless polymer localized exclusively where the probe and target sequences hybridized on the microarray. Polymer formation is visualized after a quick staining step.

Article Snippet: Positive hybridization signals on each microarray were detected by incubation with 40 μl ampliPHYTM solution (InDevR, Inc.), followed by photoactivation for approximately 1–2 min with the ampliPHOX ReaderTM and the associated ampliVIEWTM software 2.0 (InDevR, Inc.), as recommended by the manufacturer.

Techniques: Labeling, Microarray, Polymer, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: O-antigen and Virulence Profiling of Shiga Toxin-Producing Escherichia coli by a Rapid and Cost–Effective DNA Microarray Colorimetric Method

doi: 10.3389/fcimb.2012.00061

Figure Lengend Snippet: Genotyping Shiga toxin-producing Escherichia coli (STEC) with low-density DNA microarrays and the ampliPHOX colorimetric method . STEC reference strains from clinical or environmental sources with different genotypes (Table ) were analyzed by 30-mer oligonucleotide DNA microarrays and the ampliPHOX colorimetric method. The panels, shown under strain designations, illustrate polymer formation on spotted probes for microarrays hybridized with amplification reactions testing a particular STEC reference strain or controls lacking a DNA template. The array layout shows the duplicate spots with probes targeting the O-antigen and virulence genes that are represented in groups 1–4. Biotinylated probes spotted along the top, middle and bottom rows (dark gray) of the array layout correspond to positive controls for polymer formation.

Article Snippet: Positive hybridization signals on each microarray were detected by incubation with 40 μl ampliPHYTM solution (InDevR, Inc.), followed by photoactivation for approximately 1–2 min with the ampliPHOX ReaderTM and the associated ampliVIEWTM software 2.0 (InDevR, Inc.), as recommended by the manufacturer.

Techniques: Polymer, Amplification

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: O-antigen and Virulence Profiling of Shiga Toxin-Producing Escherichia coli by a Rapid and Cost–Effective DNA Microarray Colorimetric Method

doi: 10.3389/fcimb.2012.00061

Figure Lengend Snippet: Genotyping Shiga toxin-producing Escherichia coli environmental isolates with DNA microarray-based ampliPHOX colorimetric method .

Article Snippet: Positive hybridization signals on each microarray were detected by incubation with 40 μl ampliPHYTM solution (InDevR, Inc.), followed by photoactivation for approximately 1–2 min with the ampliPHOX ReaderTM and the associated ampliVIEWTM software 2.0 (InDevR, Inc.), as recommended by the manufacturer.

Techniques: Microarray, Genotyping Assay

Journal:

Article Title: Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

doi: 10.1093/nar/gkg806

Figure Lengend Snippet: Gene expression analysis of the L.tarentolae As50.1 mutant as determined by DNA microarrays and northern blot analysis. (A) Scatter plot of hybridisation intensities between TarAs50.1 (Cy5) and wild-type L.tarentolae cells (Cy3). The expression of genes represented by dots within the dashed lines are considered as similar in the two tested strains. Dashed lines indicate 2-fold differences and genes whose expression differ significantly are indicated. (B) Confirmation of DNA microarray results by northern blot analysis showing that PGPA and GSH1 were overexpressed in the same mutant. Presumably, for genes grossly overexpressed, microarray results can only be qualitative as the signals are rapidly saturated. An α-tubulin hybridisation was performed to monitor RNA and DNA loading. For each figure we show one representative of several control gels used for the various genes. For quantification see Table ​Table2.2. (C) Southern blot analysis to test whether increased RNA expression is mediated, at least in part, by gene amplification. The DNA of the parasites was digested with HindIII. 1, L.tarentolae wild-type cell; 2, TarII As50.1. Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen.

Article Snippet: Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Average fold increase compared to wild-type cells Strains Genes Microarrays Northern Southern TarAs50.1 GSH1 3.0 ± 0.2* a 10.9 12.9 PGPA 2.0 ± 0.3** b 15 3.2 TarIISbIII400.1 GSH1 11.5 ± 0.9* 44.7 3.9 (1.9 ± 0.1*) c PGPA 2.2 ± 0.3** 2.9 12.7 (6.2 ± 0.5*) c GSH2 2.8 ± 0.3* 3.6 0.8 (1.0) c SAHH 0.7 ± 0.1** 1.1 2.2(2.8 ± 0.1*) c LV39 MTX60.2 PTR1 25.3 ± 4.0* 20 17.2 MAT2 4.1 ± 0.2* 4 1.1 LV39 MTX60.4 FT 0.6* 0.12 d 0 e DHFR 2.8 ± 0.1* 11.5 7.1 Open in a separate window a * P value <0.0001. b ** P value <0.02. c Values within parenthesis correspond to gene amplification as determined using DNA microarrays. d This signal is probably coming from one of the several other FT members. e Some of the copy number of the FT family remains unchanged but other genes, indicated by asterisks, were deleted. caption a8 Correlation between microarray results, RNA expression and DNA copy number The antimonite resistant mutant L.tarentolae TarIISbIII400.1 has been described previously.

Techniques: Expressing, Mutagenesis, Northern Blot, Hybridization, Microarray, Southern Blot, RNA Expression, Amplification

Journal:

Article Title: Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

doi: 10.1093/nar/gkg806

Figure Lengend Snippet: Gene expression analysis of the L.tarentolae TarIISbIII400.1 mutant as determined by DNA microarrays and northern blot analysis. (A) Scatter plot of hybridisation intensities between Tar SbIII400.1 (Cy5) and wild-type L.tarentolae cells (Cy3). Dashed lines indicate 2-fold differences. (B) Confirmation of DNA microarray results by northern blot analysis showing that PGPA, GSH1 and GSH2 are overexpressed. An α-tubulin hybridisation was performed to monitor RNA and DNA loading. For each figure we show one representative of several control gels used for the various genes. For quantification see Table ​Table2.2. (C) Southern blot analysis to test whether increased RNA expression is mediated by gene amplification. The DNA of the parasites was digested with HindIII (blots hybridised to GSH2 and tubulin probes); EcoRI (blot hybridised to a GSH1 probe); and BamHI (blot hybridised to a PGPA probe). 1, Leishmania tarentolae wild-type cell; 2, TarIISbIII400.1. Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen.

Article Snippet: Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Average fold increase compared to wild-type cells Strains Genes Microarrays Northern Southern TarAs50.1 GSH1 3.0 ± 0.2* a 10.9 12.9 PGPA 2.0 ± 0.3** b 15 3.2 TarIISbIII400.1 GSH1 11.5 ± 0.9* 44.7 3.9 (1.9 ± 0.1*) c PGPA 2.2 ± 0.3** 2.9 12.7 (6.2 ± 0.5*) c GSH2 2.8 ± 0.3* 3.6 0.8 (1.0) c SAHH 0.7 ± 0.1** 1.1 2.2(2.8 ± 0.1*) c LV39 MTX60.2 PTR1 25.3 ± 4.0* 20 17.2 MAT2 4.1 ± 0.2* 4 1.1 LV39 MTX60.4 FT 0.6* 0.12 d 0 e DHFR 2.8 ± 0.1* 11.5 7.1 Open in a separate window a * P value <0.0001. b ** P value <0.02. c Values within parenthesis correspond to gene amplification as determined using DNA microarrays. d This signal is probably coming from one of the several other FT members. e Some of the copy number of the FT family remains unchanged but other genes, indicated by asterisks, were deleted. caption a8 Correlation between microarray results, RNA expression and DNA copy number The antimonite resistant mutant L.tarentolae TarIISbIII400.1 has been described previously.

Techniques: Expressing, Mutagenesis, Northern Blot, Hybridization, Microarray, Southern Blot, RNA Expression, Amplification

Journal:

Article Title: Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

doi: 10.1093/nar/gkg806

Figure Lengend Snippet: Gene expression analysis of the L.major LV39 MTX60.2 mutant as determined by DNA microarrays and northern blot analysis. (A) Scatter plot of hybridisation intensities between LV39 MTX60.2 (Cy5) and wild-type L.major LV39 cells (Cy3). Dashed lines indicate 2-fold differences. A number of fragments spanning ABC transporter genes cross-hybridised to the overexpressed PGPA. (B) Confirmation of DNA microarray results by northern blot analysis showing overexpression of PTR1 and MAT2. An α-tubulin hybridisation was performed to monitor RNA and DNA loading. For quantification see Table ​Table2.2. (C) Southern blot analysis to test whether increased RNA expression is mediated by gene amplification. The DNA of the parasites was digested with HindIII except for the blot hybridised to the PTR1 probe where the DNA was digested with SacI. 1, Leishmania major wild-type cell; 2, L.major LV39 MTX60.2. Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen.

Article Snippet: Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Average fold increase compared to wild-type cells Strains Genes Microarrays Northern Southern TarAs50.1 GSH1 3.0 ± 0.2* a 10.9 12.9 PGPA 2.0 ± 0.3** b 15 3.2 TarIISbIII400.1 GSH1 11.5 ± 0.9* 44.7 3.9 (1.9 ± 0.1*) c PGPA 2.2 ± 0.3** 2.9 12.7 (6.2 ± 0.5*) c GSH2 2.8 ± 0.3* 3.6 0.8 (1.0) c SAHH 0.7 ± 0.1** 1.1 2.2(2.8 ± 0.1*) c LV39 MTX60.2 PTR1 25.3 ± 4.0* 20 17.2 MAT2 4.1 ± 0.2* 4 1.1 LV39 MTX60.4 FT 0.6* 0.12 d 0 e DHFR 2.8 ± 0.1* 11.5 7.1 Open in a separate window a * P value <0.0001. b ** P value <0.02. c Values within parenthesis correspond to gene amplification as determined using DNA microarrays. d This signal is probably coming from one of the several other FT members. e Some of the copy number of the FT family remains unchanged but other genes, indicated by asterisks, were deleted. caption a8 Correlation between microarray results, RNA expression and DNA copy number The antimonite resistant mutant L.tarentolae TarIISbIII400.1 has been described previously.

Techniques: Expressing, Mutagenesis, Northern Blot, Hybridization, Microarray, Over Expression, Southern Blot, RNA Expression, Amplification

Journal:

Article Title: Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

doi: 10.1093/nar/gkg806

Figure Lengend Snippet: Gene expression analysis of the L.major LV39 MTX60.4 mutant as determined by DNA microarrays and northern blot analysis. (A) Scatter plot of hybridisation intensities between LV39 MTX 60.4 (Cy3) and wild-type L.major LV39 cells (Cy5). Dashed lines indicate 2-fold limits. (B) Confirmation of DNA microarray results by northern blot analysis showing overexpression of DHFR-TS, while the expression of one member of the FT family labelled with an asterisk is repressed. An α-tubulin hybridisation was performed for the determination of equal RNA and DNA loading. For quantification see Table ​Table2.2. (C) Southern blot analysis to test whether variation in RNA expression is mediated by variation in gene’s copy number. The DNA of the parasites was digested with SalI for the FT and α-tubulin blots and with SacI for the DHFR-TS blot. 1, Leishmania major wild-type cell; 2, L.major LV39 MTX60.4. Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. (D) Transport experiment showing a decrease in MTX accumulation in the mutant LV39 MTX60.4 cells (filled squares) compared with wild-type L.major LV39 cells (open circles).

Article Snippet: Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Average fold increase compared to wild-type cells Strains Genes Microarrays Northern Southern TarAs50.1 GSH1 3.0 ± 0.2* a 10.9 12.9 PGPA 2.0 ± 0.3** b 15 3.2 TarIISbIII400.1 GSH1 11.5 ± 0.9* 44.7 3.9 (1.9 ± 0.1*) c PGPA 2.2 ± 0.3** 2.9 12.7 (6.2 ± 0.5*) c GSH2 2.8 ± 0.3* 3.6 0.8 (1.0) c SAHH 0.7 ± 0.1** 1.1 2.2(2.8 ± 0.1*) c LV39 MTX60.2 PTR1 25.3 ± 4.0* 20 17.2 MAT2 4.1 ± 0.2* 4 1.1 LV39 MTX60.4 FT 0.6* 0.12 d 0 e DHFR 2.8 ± 0.1* 11.5 7.1 Open in a separate window a * P value <0.0001. b ** P value <0.02. c Values within parenthesis correspond to gene amplification as determined using DNA microarrays. d This signal is probably coming from one of the several other FT members. e Some of the copy number of the FT family remains unchanged but other genes, indicated by asterisks, were deleted. caption a8 Correlation between microarray results, RNA expression and DNA copy number The antimonite resistant mutant L.tarentolae TarIISbIII400.1 has been described previously.

Techniques: Expressing, Mutagenesis, Northern Blot, Hybridization, Microarray, Over Expression, Southern Blot, RNA Expression

Journal:

Article Title: Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

doi: 10.1093/nar/gkg806

Figure Lengend Snippet: Gene amplification events in L.tarentolae TarIISbIII400.1 as determined by DNA microarrays. (A) Scatter plot of hybridisation intensities between TarIISbIII400.1 and wild-type digested total DNA and labelled, respectively, with Cy5 and Cy3. This corresponds to a second generation of array with 50 genes each spotted 12 times. Dots outside the dotted lines representing 2-fold differences suggest changes in copy number in the mutant. (B) Northern blot analysis of the SAHH gene. 1, Leishmania tarentolae wild-type cell; 2, TarIISbIII400.1. Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. (C) The amplification of the SAHH gene in this mutant was confirmed by Southern blot of DNA digested with HindIII, while the other amplification events are shown in Figure ​Figure33.

Article Snippet: Sizes were determined using the 1 kb Plus DNA ladder and the 0.24–9.5 kb RNA ladder from Invitrogen. table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Average fold increase compared to wild-type cells Strains Genes Microarrays Northern Southern TarAs50.1 GSH1 3.0 ± 0.2* a 10.9 12.9 PGPA 2.0 ± 0.3** b 15 3.2 TarIISbIII400.1 GSH1 11.5 ± 0.9* 44.7 3.9 (1.9 ± 0.1*) c PGPA 2.2 ± 0.3** 2.9 12.7 (6.2 ± 0.5*) c GSH2 2.8 ± 0.3* 3.6 0.8 (1.0) c SAHH 0.7 ± 0.1** 1.1 2.2(2.8 ± 0.1*) c LV39 MTX60.2 PTR1 25.3 ± 4.0* 20 17.2 MAT2 4.1 ± 0.2* 4 1.1 LV39 MTX60.4 FT 0.6* 0.12 d 0 e DHFR 2.8 ± 0.1* 11.5 7.1 Open in a separate window a * P value <0.0001. b ** P value <0.02. c Values within parenthesis correspond to gene amplification as determined using DNA microarrays. d This signal is probably coming from one of the several other FT members. e Some of the copy number of the FT family remains unchanged but other genes, indicated by asterisks, were deleted. caption a8 Correlation between microarray results, RNA expression and DNA copy number The antimonite resistant mutant L.tarentolae TarIISbIII400.1 has been described previously.

Techniques: Amplification, Hybridization, Mutagenesis, Northern Blot, Southern Blot