Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: RNase L reduces L1 RNA accumulation in cells. ( A ) Results of qRT-PCR experiments: HeLa-M cells were co-transfected with pAD2TE1 and an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. L1 RNA levels were determined 48 h after transfection using the Sybr Green method . The X-axis indicates the RNase L co-transfected samples. The Y-axis indicates the relative expression level of L1 RNA from the transfected construct. The L1 RNA amounts were normalized with hygromycin mRNA levels (see ‘Materials and Methods’ section for detailed PCR strategy). Data are represented as the mean ± SD from three technical replicates of a single representative experiment. * P < 0.01 (Dunnett’s Multiple Comparison Test). The experiment was conducted three times (biological replicates) with similar results. ns, not significant. ( B ) Immunofluorescent Confocal Microscopy Studies: HeLa-M cells were co-transfected with pAD3TE1, a plasmid expressing a nuclear localized MS2-GFP fusion protein, and an empty vector (pcDNA 3.0) or an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy demonstrated L1 RNA accumulation in cytoplasmic foci by exploiting the 24 MS2 binding sites in pAD3TE1 L1 RNA. The top labels indicate DAPI, MS2-GFP or the antibodies used to detect the EBNA-1 and RNase L proteins. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the overlay staining. The white arrows indicate L1 cytoplasmic foci containing L1 RNA. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, SYBR Green Assay, Construct, Comparison, Confocal Microscopy, Binding Assay, Staining

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Human RNase L alone does not affect G418-resistant foci formation. ( A ) Results from the Assay: HeLa-M cells were co-transfected with pcDNA 3.0 (Gibco/Life Technologies/InVitrogen) and either an empty vector (pFLAG-CMV-2) or an amino-terminal FLAG-tagged RNase L expression plasmid. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the Assays: The X-axis depicts construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Quantification was performed as outlined in the legend to B. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. The experiment was conducted three times (biological replicates) with similar results. No statistically significant difference was found with one-way ANOVA and post hoc tests. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A) and constitutively active (NΔ385) RNase L mutant were detected from total cell lysates in western blots with anti-RNase L antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Transfection, Plasmid Preparation, Expressing, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation, Mutagenesis, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Inhibition of L1 retrotransposition by RNase L. ( A ) L1 Retrotransposition Assays: HeLa-M cells were co-transfected with pJM101/L1.3 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes amino-terminal FLAG-tagged versions of the following proteins: RNase L, A3A or RIG-I. The cells were subjected to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the L1 Retrotransposition Assays: The X-axis depicts the co-transfected construct names. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are shown as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P < 0.01 (when each test group was compared with empty vector control with Dunnett’s Multiple Comparison Test). The experiment was conducted four times (biological replicates) with similar results. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A), constitutively active (NΔ385) RNase L mutant, A3A and RIG-I proteins were detected from total cell lysates in western blots with anti-FLAG antibody 2 days after transfection. β-actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Inhibition, Transfection, Plasmid Preparation, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation, Control, Comparison, Expressing, Mutagenesis, Western Blot

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Expression of RNase L inhibits L1 retrotransposition in an EGFP -based retrotransposition assay. ( A ) Results from the assays: HeLa-M cells were co-transfected with an expression construct containing an active human L1 (pLRE3- mEGFPI ) and an empty vector (pFLAG-CMV-2), a plasmid encoding an amino-terminal FLAG-tagged RNase L expression plasmid or an amino-terminal HA-tagged A3A expression plasmid. Experiments with a retrotransposition-defective L1 pJM111-LRE3- mEGFPI served as a negative control. The cells were subjected to puromycin selection for 4 days after transfection. Fluorescence Activated Cell Sorting (FACS) was then used to screen for EGFP-positive cells. The X-axis indicates the construct name. The Y-axis indicates the percentage of EGFP-positive cells. For each sample, 2 × 10 5 cells were analyzed and the percentage of EGFP-positive cells was calculated with using the FlowJo software package. Data were analyzed with one-way ANOVA with post hoc tests and are shown as mean ± SD from a single experiment with three technical replicates. * P < 0.01 (Dunnett’s Multiple Comparison Test). The experiment was conducted four times (biological replicates) with similar results. ( B ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A), and constitutively active (NΔ385) RNase L mutants were detected in total cell lysates by western blot with anti-RNase L antibody 2 days after transfection. The A3A protein was detected using an anti-HA antibody. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Negative Control, Selection, Fluorescence, FACS, Software, Comparison, Mutagenesis, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Inhibition of IAP retrotransposition by RNase L. ( A ) IAP Retrotransposition Assays: HeLa-M cells were co-transfected with a mouse IAP expression construct (pDJ33/440N1 neo TNF ) and either an empty vector (pFLAG-CMV-2) or an expression plasmid that encodes an amino-terminal FLAG-tagged version of the following proteins: WT RNase L, a catalytically inactive RNase L mutant (R667A), a constitutively active RNase L mutant (NΔ385), A3A or RIG-I. The cells were subject to selection for 10 days and G418-resistant foci were fixed and stained with crystal violet for visualization purposes. A representative tissue culture dish for each condition is shown. ( B ) Quantitation of the IAP Retrotransposition Assays: The X-axis depicts names of constructs co-transfected into cells with the IAP construct. The Y-axis depicts the number of G418-resistant foci per cell culture dish. Data are represented as the mean ± standard deviation (SD) from a single experiment with three technical replicates. * P < 0.01 (when each test group was compared with empty vector control with Dunnett’s Multiple Comparison Test). The error bar in the Empty Vector bar is too small to visualize. The experiment was conducted three times (biological replicates) with similar results. ( C ) Protein expression analyses: The WT RNase L, catalytically inactive RNase L mutant (R667A), constitutively active RNase L mutant (NΔ385), A3A and RIG-I proteins were detected in total cell lysates by western blotting with anti-FLAG antibody 2 days after transfection. β-Actin served as loading and transfer control. Size standards are indicated in kDa at the left of the gel.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Inhibition, Transfection, Expressing, Construct, Plasmid Preparation, Mutagenesis, Selection, Staining, Quantitation Assay, Cell Culture, Standard Deviation, Control, Comparison, Western Blot

Journal: Nucleic Acids Research

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

doi: 10.1093/nar/gkt1308

Figure Lengend Snippet: Expression of RNase L blocks L1 RNP formation. HeLa-M cells were co-transfected with pES2TE1 and either an empty vector (pcDNA 3.0) or a plasmid that encodes an amino-terminal Myc-tagged RNase L expression plasmid. Immunofluorescent confocal microscopy was used to examine L1 ORF2p accumulation in cytoplasmic foci by exploiting the FLAG-HA epitope-tag in pES2TE1 48 h after transfection. The top labels indicate the antibodies used to detect the indicated proteins: anti-HA-ORF2p, red; anti-EBNA-1, green; anti-Myc RNase L, magenta. The labels on the left side of the figure indicate the empty vector or RNase L constructs that were co-transfected into cells. The rightmost column indicates the merged overlay staining. L1 ORF2p formed discrete cytoplasmic punctate localization in co-transfection experiments performed with the empty vector and RNase L catalytically inactive mutant (R667A), but not with WT RNase L. For each condition, either two or three slides were examined per experiment. About 200 cells were examined per slide and representative images were captured. The experiment was conducted three times (biological replicates) with similar results.

Article Snippet: While the HeLa-M cells used in this study are deficient in RNase L, it should be noted that other types of HeLa cells (including ATCC CCL-2 and S3) express normal levels of endogenous RNase L ( , ).

Techniques: Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Construct, Staining, Cotransfection, Mutagenesis

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