Journal:

Article Title: Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

doi:

Figure Lengend Snippet: Inducible expression of NS5A reduces IFN sensitivity of viral mRNA translation. (A) Inducible expression of NS5A 1B in HeLa 1B cells. Extracts (50 μg) prepared from Tet− (lane 1) and Tet+ (lane 2) cultures of HeLa 1B cells were analyzed by immunoblotting using a MAb specific to NS5A. The arrowhead denotes the position of NS5A 1B. We confirmed that PKR was efficiently expressed in cells from Tet− and Tet+ cultures (not shown). Positions of molecular mass standards are indicated in kilodaltons. (B) Expression of NS5A supports viral mRNA translation in IFN-treated HeLa 1B cells infected with VSV. Viral protein synthesis in cells treated with increasing concentrations of IFN was determined by biosynthetic labeling and autoradiography as described in Materials and Methods. The level of each viral protein was quantitated from autoradiograms by using a Bio-Rad GS700 imaging densitometer and computer software supplied by the manufacturer. Panels show the biosynthesis of the 29-kDa VSV matrix protein (denoted by arrowheads) in the presence (NS5A 1B+; lower panel) and absence (NS5A 1B−; upper panel) of NS5A 1B expression. The far-left lane of each panel shows an extract prepared from uninfected, untreated control cultures. Lanes represent IFN concentrations of 0 (lanes 1 and 2), 50 (lane 3), 100 (lane 4), 150 (lane 5), 200 (lane 6), 300 (lane 7), 400 (lane 8), 500 (lane 9), 600 (lane 10), 750 (lane 11), and 1,000 (lane 12) U/ml. (C) The relative level of matrix protein translation for each sample was determined by first subtracting the optical density (within the region indicated for mock-infected extracts by brackets in panel B) from that obtained for each subsequent lane. Data are presented as a ratio of VSV matrix protein translation in cells expressing NS5A to that observed in cells not expressing NS5A, for each concentration of IFN. The value of each ratio is shown above the corresponding bar.

Article Snippet: HeLa S3 Tet-Off cells (Clontech) were transfected with pTRE-NS5A 1B or pTRE-NS5A 1B-5 and selected in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), l -glutamine (200 μg/ml), G418 (100 μg/ml), hygomycin B (100 μg/ml), and Tet (2 μg/ml).

Techniques: Expressing, Western Blot, Infection, Labeling, Autoradiography, Imaging, Software, Concentration Assay

Journal:

Article Title: Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

doi:

Figure Lengend Snippet: ISDR mutations within the PKR-binding domain of NS5A confer IFN sensitivity to viral mRNA translation. (A) Inducible expression of NS5A 1B and NS5A 1B-5 in HeLa S3 cells. Extracts (50 μg) prepared from Tet− (lanes 1 and 3) and Tet+ (lane 2 and 4) cultures of HeLa 1B (lanes 1 and 2) and HeLa 1B-5 (lanes 3 and 4) cell lines were analyzed by immunoblotting using a MAb specific to NS5A. Arrowheads denote the positions NS5A, which migrates on SDS-PAGE as hypo- and hyperphosphorylated isoforms (60). We confirmed that PKR was efficiently expressed in both cell lines in the presence and absence of Tet (not shown). Positions of molecular mass standards are indicated in kilodaltons. (B) Expression of NS5A 1B, but not NS5A 1B-5, supports viral mRNA translation in IFN-treated HeLa cell lines infected with VSV. Viral protein synthesis in HeLa 1B (upper panel) and HeLa 1B-5 (lower panel) cell lines, cultured in the absence of Tet and treated with increasing concentrations of IFN, was determined by biosynthetic labeling and autoradiography as described in Materials and Methods. The level of each viral protein was quantitated as described for Fig. ​Fig.2.2. Panels show the biosynthesis of the 29-kDa VSV matrix protein (denoted by arrowheads) in the presence NS5A 1B and NS5A 1B-5 (upper and lower panels, respectively). The far-left lane of each panel shows an extract prepared from uninfected, untreated control cultures. Lanes represent IFN concentrations of 0 (lanes 1 and 2), 50 (lane 3), 100 (lane 4), 150 (lane 5), 200 (lane 6), 300 (lane 7), 400 (lane 8), 500 (lane 9), 600 (lane 10), 750 (lane 11), and 1,000 (lane 12) U/ml. (C) Translation ratios. The relative level of matrix protein translation for each sample was determined as for Fig. ​Fig.2.2. Data are presented as a ratio of VSV matrix protein translation in cells expressing NS5A 1B to that observed in cells expressing NS5A 1B-5, for each concentration of IFN. The value of each ratio is shown above the corresponding bar.

Article Snippet: HeLa S3 Tet-Off cells (Clontech) were transfected with pTRE-NS5A 1B or pTRE-NS5A 1B-5 and selected in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), l -glutamine (200 μg/ml), G418 (100 μg/ml), hygomycin B (100 μg/ml), and Tet (2 μg/ml).

Techniques: Binding Assay, Expressing, Western Blot, SDS Page, Infection, Cell Culture, Labeling, Autoradiography, Concentration Assay

Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: Increased transfection efficiency by acidic DNA compaction. PEI and pcDNA4-TO-EGFP were mixed in HBS (pH 7.4) or LBS (pH 3.5) [PEI/DNA ratio = 5 (μg/μg)], and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. At 24 h posttransfection, EGFP expression was analyzed by flow cytometry. a Representative histograms of control (shaded areas) and transfected cells (solid line) are shown. Two gates indicate EGFP expression at high levels (thick arrows) and at low to high levels (thin arrows). Transfection efficiencies were quantitated by counting the number of cells expressing EGFP at high levels and at low to high levels. b HeLa cells transiently transfected with the polyplex formed in HBS (pH 7.4) or LBS (pH 3.5) were stained with PI and analyzed for PI-stained dead cells by flow cytometry. Data represent means ± SD (n = 3)

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Expressing, Flow Cytometry, Control, Staining

Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: Effects of PEI/DNA ratios and pH of compaction medium on transfection efficiency. a, b PEI and pcDNA4-TO-EGFP were mixed in LBS (pH 3.5) at various PEI/DNA ratios (μg/μg), and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. a At 24 h posttransfection, cells were stained with PI for dead cells, and analyzed by flow cytometry for EGFP expression (filled bars) and cytotoxicity (open bars). Data represent means ± SD (n = 3). b PEI and pcDNA4-TO-EGFP were mixed in LBS (pH 3.5) at a ratio of 6 (μg/μg). Representative histograms of control (shaded areas) and transfected cells (solid line) are shown. Two gates indicate EGFP expression at high levels (thick arrows) and at low to high levels (thin arrows). c PEI and pcDNA4-TO-EGFP were mixed at a ratio of 5 (μg/μg) in HBS (pH 7.4) or LBS (pH 3.5, 4.0, 4.5), and the resulting PEI/DNA polyplex was transiently transfected into HeLa cells. Transfection efficiencies were quantitated by counting the number of cells expressing EGFP at high levels (filled bars) and at low to high levels (open bars). Results are expressed as values relative to the number of cells expressing EGFP using HBS as DNA compaction medium (pH 7.4). Data represent means ± SD (n = 3). Transfection efficiencies were 2.5 and 16.5% at pH 7.4 and pH 4.0, respectively in cells expressing EGFP at high levels. d, e Five microgram of new or old PEI and 1 μg of pcDNA4-TO-EGFP were mixed in LBS (pH 4.0), and 5 μg of new PEI and 5 μg of old PEI (mix) and 1 μg of pcDNA4-TO-EGFP were mixed in LBS (pH 4.0). The resulting PEI/DNA polyplex was transiently transfected into HeLa S3 cells, and transfection efficiencies were quantitated by counting the number of cells expressing EGFP. Data represent means ± SD (n = 3)

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Staining, Flow Cytometry, Expressing, Control

Journal: Cytotechnology

Article Title: Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine

doi: 10.1007/s10616-010-9259-z

Figure Lengend Snippet: Transfection into COS-1, HeLa S3, Dami, and HCT116 cells. a–c EGFP expression was examined by fluorescence microscopy. Scale bars, 10 μm. a COS-1 and HeLa S3 cells were transiently transfected with the polyplex formed by mixing 5 μg of pcDNA4-TO-EGFP with 25 μg of PEI in LBS (pH 4.0), and after 12 h of culture the medium was replaced with fresh serum-containing medium. b COS-1 cells were transiently transfected with the polyplex formed by mixing 1 μg of pcDNA4-TO-EGFP with 5 μg of PEI in LBS (pH 4.0) or an optimal amount of TransIT-LT1. c Dami cells in suspension culture were attached to culture dishes, as described under “Materials and methods”, and transiently transfected with the polyplex formed by mixing 4 μg of pcDNA4-TO-EGFP with 26 μg of PEI in LBS (pH 4.0). d HCT116 cells were transiently transfected once or twice with the polyplex formed by mixing 6 μg of pCAG/TetR-IRES-CD25 with 7.5 μg of PEI in LBS (pH 4.0), as described under “Materials and methods”, and stained with anti-CD25 antibody. Representative histograms of control (shaded areas) and transfected cells (solid line) are shown for CD25 expression at low to high levels (thin arrows). e HeLa S3 cells were stably transfected with the polyplex formed by mixing pcDNA4-TO-EGFP with PEI in LBS, as described under “Materials and methods”. Zeocin-resistant colonies were cloned in 2 weeks and observed by fluorescence microscopy. Scale bar, 10 μm

Article Snippet: Cell lines Human epithelial HeLa and HeLa S3 cells (Japanese Collection of Research Bioresources, Osaka) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal bovine serum (FBS) and with 5% bovine serum (BS), respectively.

Techniques: Transfection, Expressing, Fluorescence, Microscopy, Suspension, Staining, Control, Stable Transfection, Clone Assay