apo ar  (Thermo Fisher)

Journal: Virology

Article Title: The TP53-Induced Glycolysis and Apoptosis Regulator mediates cooperation between HTLV-1 p30 II and the retroviral oncoproteins Tax and HBZ and is highly expressed in an in vivo xenograft model of HTLV-1-induced lymphoma

doi: 10.1016/j.virol.2018.05.007

Figure Lengend Snippet: The HTLV-1 p30II protein suppresses Tax/HBZ-induced ROS production through the activation of TIGAR. (A) Jurkat T-cells were transfected with either an empty CβS vector, or expression constructs for HTLV-1 Tax or HBZ, and then stained with the JC-1 dye (Molecular Probes) to detect changes in mitochondrial membrane potential. The JC-1 dye forms aggregates on regions of high mitochondrial polarization (red signal), whereas depolarized regions associated with oxidative damage are indicated by green-fluorescence. The chemical uncoupler, CCCP (Sigma-Aldrich), was included as a positive control to induce mitochondrial membrane depolarization. The JC-1-associated red and green fluorescent signals were quantified by flow cytometry. (B) Graph depicting the relative JC-1 red, green, and red/green (yellow) fluorescent signals in transfected Jurkat cells as shown in A. The data represent the mean +/- standard deviation (error bars) from three independent experiments. (C) HT-1080 fibrosarcoma cells were transfected with an empty CβS vector, or expression constructs for HTLV-1 Tax or HBZ (Myc-tagged), and confocal microscopy was performed to visualize JC-1 red and green fluorescence associated with alterations in mitochondrial membrane polarization. CCCP was included as a positive control. (D and E) The expression of the HTLV-1 Tax and HBZ proteins in transfected Jurkat T-cells (D) and HT-1080 cells (E) was detected by immunoblotting. Relative Actin levels are shown for comparison. (F) The accumulation of intracellular ROS in HT-1080 cells cotransfected with either an empty CβS vector, or expression constructs for HTLV-1 Tax, HBZ and/or p30II (HA-tagged), was detected by staining the cells with the fluorescent chemical ROS probe, CM-H2DCFDA (Molecular Probes), and the percentages of CM-H2DCFDA-positive cells were quantified using fluorescence-microscopy, as compared to the total numbers of cells visualized with a DIC phase-contrast filter under a 20x objective lens (see micrographs in Supplemental Fig. S1B). (G) To determine if the activation of TIGAR contributes to the suppression of Tax/HBZ-induced ROS production by p30II, the cells were cotransfected as in F and then repeatedly transfected with a siRNA-tigar oligonucleotide or scrambled RNA (scrRNA) negative control. A pcDNA3.1-FLAG-tagged-TIGAR expression construct was included in some samples to determine the effects of overexpressing the TIGAR protein. The samples were then stained with CM-H2DCFDA and the relative percentages of CM-H2DCFDA-positive cells were determined using fluorescence-microscopy as in F. (H) The specificity of the siRNA-tigar oligonucleotide to inhibit TIGAR expression was determined by transfecting HT-1080 cells with siRNA-tigar, a scrRNA control, or empty CβS vector and then immunoblotting to detect the endogenous TIGAR protein. Alternatively, the cells were cotransfected with pcDNA3.1-FLAG-tagged-TIGAR and either an empty CβS vector, siRNA-tigar, or a scrRNA control and the FLAG-tagged TIGAR protein was detected by immunoblotting using a monoclonal Anti-FLAG M2 primary antibody. Relative Actin levels are shown for comparison. All the data is representative of at least three independent experiments. The data in B, F, and G represent the mean of the experiments ± standard deviation (error bars).

Article Snippet: The visualization and quantification of ROS-positive cells and oncogenic foci-formation by HTLV-1 Tax, HBZ, and p30 II -GFP in cotransfected cells were carried out using a Nikon Eclipse TE2000-U inverted microscope and D-Eclipse C1 confocal imaging system equipped with 633 nm and 543 nm He/Ne and 488 nm Ar lasers and a Plan-Apo 20x/0.75 objective lens.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Staining, Fluorescence, Positive Control, Flow Cytometry, Standard Deviation, Confocal Microscopy, Western Blot, Microscopy, Negative Control

Journal: Virology

Article Title: The TP53-Induced Glycolysis and Apoptosis Regulator mediates cooperation between HTLV-1 p30 II and the retroviral oncoproteins Tax and HBZ and is highly expressed in an in vivo xenograft model of HTLV-1-induced lymphoma

doi: 10.1016/j.virol.2018.05.007

Figure Lengend Snippet: The HTLV-1 p30II protein inhibits Tax/HBZ-induced cellular senescence and cooperates with these major viral oncoproteins to induce oncogenic foci-formation in vitro. (A and B) To determine if p30II prevents Tax/HBZ-induced cellular senescence in HeLa cells as reported in Ho et al., 2012 and Kuo and Giam, 2006, the cells were cotransfected with an empty CβS vector or expression constructs for HTLV-1 Tax, HBZ, and/or p30II-GFP in various combinations. The cells were then cultured for five days and subsequently stained using an X-Gal solution to detect senescence-associated Beta-galactosidase (SA-β-gal) expression (blue signal in micrographs). DIC phase-contrast is included in the merged images for reference. The data in A represent the mean ± standard deviation (error bars) from three independent experiments. (C) The expression of the HTLV-1 Tax, p30II-GFP, and HBZ (Myc-tagged) proteins was detected by SDS-PAGE and immunoblotting. The relative levels of Actin are shown for comparison. (D) The parental HT-1080 cells and transiently-amplified HT-1080/HTLV-1 ACH.wt and ACH.p30II mutant proviral clones were cotransfected with an empty CβS vector, or expression constructs for HTLV-1 p30II-GFP or TIGAR (FLAG-tagged). Alternatively, the HT-1080/ACH.p30II mutant cells were transduced with lentiviral-HTLV-1 p30II (HA) particles or an empty pLenti vector. Certain samples were also repeatedly transfected with a siRNA-tigar oligonucleotide or scrRNA negative control. The cells were then stained with an X-Gal solution to detect SA-β-gal expression and analyzed by microscopy using a 20x objective lens to quantify the numbers of senescent cells per well. The data in D represent the mean ± standard deviation (error bars) from three independent experiments. (E-H) To determine whether the HTLV-1 p30II protein cooperates with the viral oncoproteins Tax and HBZ, the HFL1 human fibroblast cell-line (a pseudodiploid cell-line with a stable karyotype which grows as a uniform monolayer) was cotransfected with an empty CβS vector, or expression constructs for HTLV-1 Tax, HBZ, or p30II-GFP in various combinations. The stably transfected cells were monitored over a three-week period for the formation of oncogenically-transformed colonies (i.e., loss of contact-inhibition; see DIC phase-contrast images in H). The transformed colonies were fixed and stained with a methylene blue solution; and the numbers of transformed colonies per well were quantified by microscopy using a 10x objective. The expression of HTLV-1 p30II-GFP within transformed colonies was visualized by direct fluorescence-microscopy (top panels in H). The data in E, F, and G represent the mean ± standard deviation (error bars) from three independent experiments.

Article Snippet: The visualization and quantification of ROS-positive cells and oncogenic foci-formation by HTLV-1 Tax, HBZ, and p30 II -GFP in cotransfected cells were carried out using a Nikon Eclipse TE2000-U inverted microscope and D-Eclipse C1 confocal imaging system equipped with 633 nm and 543 nm He/Ne and 488 nm Ar lasers and a Plan-Apo 20x/0.75 objective lens.

Techniques: In Vitro, Plasmid Preparation, Expressing, Construct, Cell Culture, Staining, Standard Deviation, SDS Page, Western Blot, Amplification, Mutagenesis, Clone Assay, Transduction, Transfection, Negative Control, Microscopy, Stable Transfection, Transformation Assay, Inhibition, Fluorescence

Journal: PLoS ONE

Article Title: Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression

doi: 10.1371/journal.pone.0204595

Figure Lengend Snippet: A) Dissociated adhered islets were treated for 14 days under low glucose (5 mM) conditions with recombinant proteins identified through mass spectrometry of functional fractions. Only ApoE significantly upregulated Ins2 and Ucn3 expression controls. B) Dissociated adhered islets were cultured for a period of 14 days under low glucose (5 mM) conditions and either treated with or without ApoE. ApoE significantly increased the expression of key beta cell markers including Ucn3 , Ins2 , Mafa , Nkx6 . 1 , Pcsk1 , Sur1 and Glut2 . Notably, increase in the expression of Nkx2 . 2 was also observed although not significant. C) Whole adhered pancreatic islets were cultured for a period of 14 days under low glucose (5 mM) conditions with or without ApoE. ApoE treatment did not significantly increase β-cell gene expression relative to controls until D14. Glut2 , Nk6 . 1 , Nkx2 . 2 and Ins2 did not show significant increases relative to controls until D14 (n = 4). D) Whole rat pancreatic islets were cultured for a period of 14 days with and without ApoE. Glucose stimulated insulin secretion (GSIS) was measured by providing a low glucose (2.8 mM) or high glucose (16.8 mM) challenge and measuring insulin secretion by ELISA. Secreted insulin concentrations were normalized to islet DNA content. ApoE-treated islets showed no significant changes in insulin release upon glucose stimulation compared to controls (n = 4). E) GSIS data for rat pancreatic islets cultured in large non-sticky flasks in suspension for a period of 7 days under high glucose (11 mM) conditions with and without ApoE. ApoE-treated islets show comparable levels of insulin release as controls by day 7 (n = 6). Data presented as mean ± SEM, where p<0.05 was considered statistically significant.

Article Snippet: Cells were treated with either 1–2 μg/mL human recombinant ApoE (R&D systems), 20 uM JAK2 inhibitor (Tyrphostin AG490, Sigma), 2.5 uM STAT3 inhibitor (Pp-YLKTK-mts, Millipore), 20 ug LDL Receptor Blocking Peptide (Cayman Chemical), 50 mg of Cholesterol in 1 g of methyl β-cyclodextrin (Sigma), 22R-Hydroxycholesterol 10 uM (Sigma), Simvastatin 1uM (Sigma), 2 units/ml of Heparinase III (Sigma) and 500 uM Palmitate (Sigma).

Techniques: Recombinant, Mass Spectrometry, Functional Assay, Expressing, Cell Culture, Gene Expression, Enzyme-linked Immunosorbent Assay, Suspension

Journal: PLoS ONE

Article Title: Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression

doi: 10.1371/journal.pone.0204595

Figure Lengend Snippet: A) Gene expression analysis of pancreatic whole islets in adhered culture for 14 days with ApoE as well as an LDL receptor blocking peptide, Heparinase and high glucose (16.7 mM) with 500 uM (GLT). ApoE significantly increased the expression of key beta cell markers when cultured with the LDL receptor blocking peptide group. In contrast, the heparinase treated group showed no significant changes in gene expression between control and ApoE treated islets. Similarly, GLT conditions also abolished the effect of ApoE on maintaining the β-cell gene expression profile (n = 3–4), in contrast to the ApoE D14 group. B) Gene expression analysis of pancreatic whole islets in adhered culture for 14 days with ApoE as well as cyclodextrin loaded with cholesterol, 22R-Hydroxycholesterol, Simvastatin and Polylysine. High levels of cholesterol abolished the effect of ApoE on maintaining the β-cell gene expression profile, while LXR activation by 22R-Hydroxycholesterol enhanced β-cell gene expression similar to ApoE. Neither Simvastatin nor Polylysine alone had a significant effect on β-cell gene expression, (n = 3–4). C) Viability assay of islets showing no significant changes between the number of viable and dead cells in both control and ApoE-treated islets (n = 10). Scale bar, 50 uM. Data presented as mean ± SEM, where p<0.05 was considered statistically significant and n.s. means not significant.

Article Snippet: Cells were treated with either 1–2 μg/mL human recombinant ApoE (R&D systems), 20 uM JAK2 inhibitor (Tyrphostin AG490, Sigma), 2.5 uM STAT3 inhibitor (Pp-YLKTK-mts, Millipore), 20 ug LDL Receptor Blocking Peptide (Cayman Chemical), 50 mg of Cholesterol in 1 g of methyl β-cyclodextrin (Sigma), 22R-Hydroxycholesterol 10 uM (Sigma), Simvastatin 1uM (Sigma), 2 units/ml of Heparinase III (Sigma) and 500 uM Palmitate (Sigma).

Techniques: Gene Expression, Blocking Assay, Expressing, Cell Culture, Control, Activation Assay, Viability Assay

Journal: PLoS ONE

Article Title: Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression

doi: 10.1371/journal.pone.0204595

Figure Lengend Snippet: A) List of upregulated proteins from the phospho explorer array from ApoE-treated whole islets in adherence compared to controls. Fold changes are considered significant when they are greater than 1.5. The results of the Antibody Array Assay identified STAT3 (phosphorylated at Ser727) as the most-upregulated protein. JAK2 (phosphorylated at Tyr221) was also among the most upregulated proteins. B) Left, Western blot analysis of phosphorylated STAT3 and JAK2, confirming the array results showing a nearly two-fold increase in the ApoE treated islets at day 14. Right, quantification of bands using Image J software. C) Western blot analysis using STAT3 and JAK2 inhibitors, which demonstrates that these inhibitors can inhibit the phosphorylation of these proteins in whole adhered islets at day 14 with or without ApoE. D) Gene expression analysis of whole adhered pancreatic islets cultured for 14 days with ApoE as well as STAT3 and Jak2 inhibitors. As expected, ApoE treatment significantly increased β-cell gene expression relative to controls, while this increase was abolished in islets treated with STAT3 and JAK2 inhibitors. Data presented as mean ± SEM, where p<0.05 was considered statistically significant (n = 4).

Article Snippet: Cells were treated with either 1–2 μg/mL human recombinant ApoE (R&D systems), 20 uM JAK2 inhibitor (Tyrphostin AG490, Sigma), 2.5 uM STAT3 inhibitor (Pp-YLKTK-mts, Millipore), 20 ug LDL Receptor Blocking Peptide (Cayman Chemical), 50 mg of Cholesterol in 1 g of methyl β-cyclodextrin (Sigma), 22R-Hydroxycholesterol 10 uM (Sigma), Simvastatin 1uM (Sigma), 2 units/ml of Heparinase III (Sigma) and 500 uM Palmitate (Sigma).

Techniques: Ab Array, Western Blot, Software, Phospho-proteomics, Gene Expression, Cell Culture