Journal: The Journal of Biological Chemistry

Article Title: Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9

doi: 10.1074/jbc.RA118.002890

Figure Lengend Snippet: Rpl29 methylation shows no effect on global protein synthesis. A, Western blotting showing the absence of Rpl29 in Rpl29−/− NIH 3T3 clones generated by CRISPR-Cas9. B, exogenous Rpl29 or the K5R mutant (untagged) was stably expressed in Rpl29−/− NIH 3T3 cells, and the cell lysates were immunoblotted with Rpl29 antibody and D8T9P, respectively. C, SUnSET assay showing global protein synthesis in the indicated cell lines. As a control for protein synthesis inhibition, NIH 3T3 cells were treated with cycloheximide (CHX, 100 μg/ml) for 12 h before the addition of puromycin. D, [35S]methionine labeling assay showing global protein synthesis in the indicated cell lines. Shown are the mean ± S.D. from three independent experiments. The significance of differences was determined with one-way analysis of variance (ANOVA). *, p < 0.05; ns, not significant. E, polysome profiling showing similar fractions of mRNPs and polysomes in all cell lines.

Article Snippet: J1 mESCs or NIH 3T3 cells were transiently co-transfected with the pCAG-Cas9-IRES-GFP vector and two synthesized gBlocks (Integrated DNA Technologies) containing the U6 promoter and Rpl29 targeting sequences in intron 1 and exon 4, respectively ( Fig. S1 A ).

Techniques: Methylation, Western Blot, Clone Assay, Generated, CRISPR, Mutagenesis, Stable Transfection, Inhibition, Labeling

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: Down-regulation of MOV10 (Moloney Leukemia Virus 10) enhances Hepatitis B virus (HBV) gene expression in HepAD38 cells. ( A ) Time course of the experiment. HepAD38 cells were transfected with 50 nM siMOV10 or siControl 24 h before tetracycline (tet) removal. Cells were collected two days after tet removal. ( B ) Western blot showing HBV core and MOV10 expression in siRNA treated cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( C ) Quantification of HBV core expression determined by western blotting. ( D ) Intracellular HBV pgRNA level determined by RT-qPCR. Data were normalized to GAPDH mRNA expression. ( E ) Intracellular HBV DNA level determined by qPCR. Data represent mean ± standard deviation from two independent experiments. * p -value < 0.05, two-tailed unpaired t -test.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: Down-regulation of MOV10 enhances HBV gene expression in HepG2 cells. ( A ) Time course of the experiment. HepG2cells were transfected with 20 nM siMOV10 or siControl 24 h before transfection with pHBV plasmid. Cells were collected two days after HBV plasmid transfection. ( B ) Western blot showing HBV core and MOV10 expression in siRNA treated cells. GAPDH was used as a loading control. ( C ) Quantification of HBV core expression determined by western blotting. ( D ) Intracellular HBV pgRNA level determined by RT-qPCR. Data were normalized to GAPDH mRNA expression. ( E ) Intracellular HBV DNA level determined by qPCR. Data represent mean ± standard deviation from two independent experiments. * p -value < 0.05, two-tailed unpaired t -test.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: HBV gene expression in HepG2 MOV10-KO cells. ( A ) Western blot showing HBV core and MOV10 in HepG2 MOV10-KO cells compared to HepG2 parent cells. GAPDH was used as a loading control. ( B ) Quantification of HBV core expression determined by western blotting. ( C ) Intracellular HBV pgRNA level determined by qPCR after cDNA synthesis. Data were normalized to RPL18A mRNA expression. Data represent mean ± standard deviation from two independent experiments. * p -value < 0.05, two-tailed unpaired t -test.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: Down-regulation of MOV10 enhances HBV infection of HepG2-sodium taurocholate co-transporting polypeptide (NTCP) cells. HepG2-NTCP-C7 stable cell lines, shControl, and shMOV10, were infected with 250 HBV genome equivalents per cell. Five days later, cells were collected. ( A ) Western blotting showing MOV10 expression in shMOV10 stable cell line compared to shControl. GAPDH was used as a loading control. ( B ) Intracellular HBV pgRNA level determined by RT-qPCR. Data were normalized to GAPDH mRNA expression. ( C ) Intracellular HBV DNA level determined by qPCR. Data represent mean ± standard deviation from at least two independent experiments. * p -value < 0.05, two-tailed unpaired t -test.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Infection, Stable Transfection, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation, Two Tailed Test

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: MOV10 over-expression diminishes HBV infection of HepG2-NTCP cells. HepG2-NTCP-3E8 parent cells and HepG2-NTCP-3E8 HA-MOV10 cells were infected with 400 HBV genome equivalents per cell. Five days later, cells were collected. ( A ) Western blot showing MOV10 expression in 3E8 HA-MOV10 stable cell line compared to HepG2-NTCP-3E8 parent cells. GAPDH was used as a loading control ( B ) Intracellular HBV pgRNA level determined by RT-qPCR. Data were normalized to GAPDH mRNA. ( C ) Intracellular HBV DNA level determined by qPCR. Data represent mean ± standard deviation from two independent experiments. * p -value < 0.05.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Over Expression, Infection, Western Blot, Expressing, Stable Transfection, Quantitative RT-PCR, Standard Deviation

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: HBV pre-genomic RNA co-immunoprecipitated with MOV10. ( A ) HepG2-NTCP clone 7 cells were infected with HBV and harvested after five days. Immunoprecipitation was performed with an antibody against human MOV10 in the HBV-infected cells and in the non-infected control cells. SYBR Green-based RT-qPCR was done using primers against HBV pre-genomic RNA. ( B ) Human GAPDH mRNA co-immunoprecipitated with MOV10. SYBR Green-based RT-qPCR was done using primers against human GAPDH mRNA. ( n = two independent experiments, ** p = 0.0015, **** p < 0.0001, two-way ANOVA Sidak’s multiple comparisons test).

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Immunoprecipitation, Infection, SYBR Green Assay, Quantitative RT-PCR

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: No specific interaction between MOV10 and HBV core protein. 293T cells were transfected with pC183 and peYFP-MOV10, pC183 and peYFP-empty, peYFP-MOV10 and peYFP-empty, or peYFP-empty only. Where indicated, lysates were pre-treated with RNase A prior to immunoprecipitation with the indicated antibodies. Immunoprecipitates were analyzed by western blotting using the indicated antibodies. ( A ) Proteins in lysates were immunoprecipitated with the anti-HBV core antibody. Immunoprecipitates were analyzed by western blotting using the anti-MOV10 and anti-HBV core antibodies. ( B ) Proteins in lysates were immunoprecipitated with the anti-MOV10 antibody. Immunoprecipitates were analyzed by western blotting using anti-MOV10 and anti-HBV core antibodies. ( C ) Western blotting showing detectable levels of MOV10 and HBV core proteins in tested lysates.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Viruses

Article Title: Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

doi: 10.3390/v11070651

Figure Lengend Snippet: MOV10 over-expression does not induce the degradation of HBV pgRNA. 293T cells were transfected with HBV core-deficient plasmid (Cp-deficient pHBV) and peYFP-MOV10 or peYFP-empty (eYFP) plasmids. After 48 h, the cells were treated with 5 µg/mL Actinomycin D. The cells were harvested at 0, 2, 4, 6 h after Actinomycin D treatment. Total RNA was extracted and HBV pgRNA levels were determined relative to the 0 h time point. RPL18A mRNA was used as a normalization control. Single phase decay curves are shown. The dotted line represents cells transfected with Cp-deficient pHBV and peYFP in the absence of Actinomycin D. The solid black line represents cells transfected with Cp-deficient pHBV and peYFP in the presence of Actinomycin D. The grey line represents cells transfected with Cp-deficient pHBV and peYFP-MOV10 in the presence of Actinomycin D. Data represent mean ± standard deviation from two independent experiments.

Article Snippet: The plasmid harboring Cas9, guide-RNA (gRNA), and GFP (pCas9-GFP-Mov10_T1) was purchased from OriGene Technologies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Standard Deviation