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: Briefly, the plasmids were double digested with Bst BI and Sfo I, followed by 3′ end filling with Klenow fragment of DNA polymerase, and blunt-end cloned into the modified pcDNA 3.0 vector using T4 DNA ligase (New England BioLabs).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, SYBR Green Assay, Construct, 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: Briefly, the plasmids were double digested with Bst BI and Sfo I, followed by 3′ end filling with Klenow fragment of DNA polymerase, and blunt-end cloned into the modified pcDNA 3.0 vector using T4 DNA ligase (New England BioLabs).

Techniques: Transfection, Plasmid Preparation, Expressing, Selection, Staining, Quantitation Assay, Construct, Cell Culture, Standard Deviation, 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 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: Briefly, the plasmids were double digested with Bst BI and Sfo I, followed by 3′ end filling with Klenow fragment of DNA polymerase, and blunt-end cloned into the modified pcDNA 3.0 vector using T4 DNA ligase (New England BioLabs).

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