Journal: Frontiers in Plant Science

Article Title: CRISPR-Cas9: Tool for Qualitative and Quantitative Plant Genome Editing

doi: 10.3389/fpls.2016.01740

Figure Lengend Snippet: Specific commercial products and services available to the researchers to implement CRISPR technology.

Article Snippet: Integrated DNA technologies (IDT) , Human HPRT PCR Primer Mix, Mouse HPRT PCR Primer Mix, Nuclease Free Duplex Buffer , S.p. Cas9 Expression Plasmid , S.p. Cas9 Nuclease 3NLS (100, 500 μg) , CRISPR Negative Control crRNA, CRISPR Positive Control crRNA.

Techniques: CRISPR, Genome Wide, Clone Assay, Stable Transfection, Selection, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Negative Control, Positive Control, Construct, Knock-In, Multiplex Assay, Amplification, Sequencing

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Expression of three isoforms of endogenous Sirt2 transcripts and proteins increases upon HBV replication. (A) Expression of endogenous Sirt2 protein increases in HBV-replicating cells. Huh7 cells were mock transfected (lane 1) or transfected with 4 μg of 1.3mer HBV WT (ayw) (lane 2). Lysates were prepared at 72 h posttransfection. Prepared lysates were subjected to SDS-PAGE and immunoblotting to detect proteins or 1% native agarose gel electrophoresis followed by immunoblotting to detect core particles. Endogenous Sirt2, HBc, acetylated α-tubulin, and α-tubulin proteins were detected using rabbit polyclonal anti-Sirt2 PA3-200 (1:1,000), rabbit polyclonal anti-HBc (1:1,000) (65), mouse monoclonal anti-acetylated α-tubulin (1:1,000; T 6793; Sigma-Aldrich), and mouse monoclonal anti-α-tubulin (1:1,000; sc-8035; Santa Cruz) antibodies, respectively. Relative levels of acetylated α-tubulin and three isoforms of Sirt2 proteins were measured using ImageJ 1.46r. Tubulin was used as a loading control. To measure viral DNA synthesis, Southern blot analysis was performed. HBV DNA isolated from core particles was separated by agarose gel electrophoresis, transferred to a nylon membrane, and subjected to autoradiography after hybridization to 32P-labeled random-primed probe specific for the full-length HBV. Shown are HBV replicative intermediate, double-stranded linear, and partially double-stranded relaxed circular DNAs (HBV RI DNA, DL, and RC, respectively). (B) Comparison of anti-Sirt2 H-95 and anti-Sirt2 PA3-200 antibodies to show localization of Sirt2.5 in the nucleus and Sirt2.1 and Sirt2.2 in the cytoplasm. Endogenous Sirt2 levels in Huh7 cells were detected by anti-Sirt2 H-95 (1:300) (a) and PA3-200 (1:300) (b) antibodies. Digital images of stained cells were captured under a confocal microscope (LSM710; Zeiss, Germany). Data are representative of three independent experiments. (C) RPA to detect three transcripts of Sirt2 isoforms in HBV replicating cells. An in vitro-transcribed radiolabeled antisense RNA probe (367 nt) was hybridized overnight at 50°C with total RNA from mock-transfected and 4 μg of 1.3mer HBV WT-transfected Huh7 cells. Following RNase A/T1 (EN0551; Thermo Fisher Scientific) digestion, protected RNAs (Sirt2.1, 350 nt; Sirt2.2, 290 nt; and Sirt2.5, 180 nt) were electrophoresed in a 5% polyacrylamide–8 M urea gel and visualized by autoradiography. (D) HBV replication increases expression of Sirt2.1, Sirt2.2, and Sirt2.5 proteins. Transiently mock- and 1.3mer HBV WT-transfected (4 μg) Huh7 cells (lanes 1 and 2), HepG2 and HBV replicating stable HepG2.2.15 cells (lanes 3 and 4), and tetracycline-treated and -removed HepAD38 cells (lanes 5 and 6) were cultured for 72 h. Total lysates and cytoplasmic and nuclear fractions were prepared from whole cells by differential centrifugation (2, 69) and subjected to SDS-PAGE and immunoblotting with PA3-200 anti-Sirt2 (1:1,000) antibody. The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients. Protein lysates from biopsy specimens were prepared in a weight per volume ratio in mPER buffer. SDS-PAGE and immunoblotting were performed as described above. NT, adjacent nontumor; T, tumor. Data are presented as the means and standard deviations (SD) from three independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Agarose Gel Electrophoresis, Control, DNA Synthesis, Southern Blot, Isolation, Membrane, Autoradiography, Hybridization, Labeling, Random Primed, Comparison, Staining, Microscopy, In Vitro, Cell Culture, Centrifugation

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Overexpression of Sirt2.5 reduces HBV viral transcription and cccDNA levels. (A) Luciferase reporter assay shows reduced HBV enhancer and promoter activity upon overexpression of Sirt2.5. HepG2 (first row) and Huh7 (second row) cells were transiently transfected with 2 μg of the indicated luciferase reporter vectors in the presence of 3×FLAG-Sirt2.1 (2 μg) or 3×FLAG-Sirt2.5 (2 μg). At 72 h posttransfection, lysates were prepared (E153A; Promega) and luciferase activity was measured. Luciferase activity relative to the respective control luciferase reporter vectors is presented. (B) Northern blotting to show the reduced levels of HBV transcripts upon overexpression of Sirt2.5. HepG2 (lanes 1 to 4) and Huh7 (lanes 5 to 8) cells were transiently mock transfected (lanes 1 and 5) or transfected with 4 μg of 1.3mer HBV WT (ayw) (lanes 2 and 6), 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.1 construct (lanes 3 and 7), or 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.5 construct (lanes 4 and 8). The amount of transfected DNA was adjusted using pcDNA3. At 72 h posttransfection, total RNA was extracted for Northern blotting; 20 μg of total RNA was separated by 1.2% formaldehyde gel electrophoresis, transferred to nylon membranes, hybridized, and subjected to autoradiography as described in the legend of Fig. 1A for Southern blotting. The 3.5-kb pgRNA and the 2.1- and 2.4-kb mRNAs encoding the S protein are indicated. Ribosomal RNAs (28S and 18S rRNAs) are included as a loading control. (C) Overexpression of Sirt2.5 reduced the amounts of HBV cccDNA and viral mRNA in HBV-infected cells. HepG2-hNTCP-C9 cells transduced with pCDH (lane 2), 3×FLAG-Sirt2.1 (lane 3), or 3×FLAG-Sirt2.5 (lane 4) were plated on collagen-coated 6-well plates, mock infected (lane 1) or infected with 1.7 × 103 GEq of HBV per cell (lanes 2 to 4), and incubated for 5 days. Total RNA then was extracted and Northern blotting was performed as described above (third panel). At 9 days postinfection at 100% confluence, cccDNA was extracted through Hirt protein-free DNA extraction procedure, with minor modifications (2, 38). Southern blotting of cccDNA without linearization (top) or following linearization with EcoRI (second panel) was performed. Data are presented as the means and SD from three (A and C) or four (B) independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Over Expression, Luciferase, Reporter Assay, Activity Assay, Transfection, Control, Northern Blot, Construct, Nucleic Acid Electrophoresis, Autoradiography, Southern Blot, Infection, Transduction, Incubation, DNA Extraction

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Overexpression of Sirt2.5 decreases HBV DNA synthesis. (A) Overexpression of Sirt2.5 decreases HBV replication in transiently transfected cells. HepG2 (lanes 1 to 4) and Huh7 (lanes 5 to 8) cells were mock transfected (lanes 1 and 5) or transfected with 4 μg of 1.3mer HBV WT (ayw) (lanes 2 and 6), 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.1 construct (lanes 3 and 7), or 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.5 construct (lanes 4 and 8). Lysates were prepared at 72 h posttransfection. The amount of transfected DNA was adjusted using pcDNA3. (B) Overexpression of Sirt2.5 reduces HBV replication in HBV-infected cells. HepG2 (lane 1) and HepG2-hNTCP-C9 (lanes 2 to 5) cells were infected as described in the legend to Fig. 2. SDS-PAGE and immunoblotting of proteins, native agarose gel electrophoresis and immunoblotting of core particles, and Southern blotting of HBV DNA were performed as described in the legend to Fig. 1. The HBV hNTCP receptor was detected using a mouse anti-rhodopsin monoclonal anti-C9 (1:1,000) antibody. Relative levels of acetylated α-tubulin, Sirt2, core particles, and HBV DNA were measured using ImageJ 1.46r. Tubulin was used as a loading control. (C) Overexpression of Sirt2.5 does not affect pgRNA encapsidation in HBV-infected cells. To detect encapsidated pgRNA and total RNA from Sirt2.1- or Sirt2.5-overexpressing HBV-infected HepG2-NTCP-C9 cells, in vitro-transcribed DIG-UTP-labeled antisense RNA probe (446 nt) was hybridized overnight at 50°C with pgRNA from isolated core particles or 10 μg total RNA. Protected RNA (369 nt) following RNase digestion was run on a 5% polyacrylamide–8 M urea gel, transferred to nylon membranes, immunoblotted with anti-DIG-AP, and visualized with CSPD. The upper panel shows encapsidated pgRNA, while the lower panel depicts total RNA. Data are presented as the mean values from three independent experiments. Statistical significance was evaluated using Student's t test. **, P < 0.005; ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Over Expression, DNA Synthesis, Transfection, Construct, Infection, SDS Page, Western Blot, Agarose Gel Electrophoresis, Southern Blot, Control, In Vitro, Labeling, Isolation

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: N-terminal 40 amino acids of Sirt2 are not important for upregulation of HBV replication. (A) Schematic diagram showing Sirt2 isoforms 1, 2, and 5 and the mutants of Sirt2.5 in which the NES and/or N-terminal truncated catalytic domain were restored. Residues are numbered according to full-length Sirt2 isoform 1. The nuclear export signal (NES; dotted), the catalytic domain (CD; black), and the nuclear localization signals (NLSs; gray) are indicated. (B) Both the NES and CD may be important for cytoplasmic localization of Sirt2. HEK293T cells were mock transfected (lane 1) or transiently transfected with 4 μg of 3×FLAG-tagged Sirt2.1 (lane 2), Sirt2.2 (lane 3), Sirt2.5 (lane 4), Sirt2.5-NES (lane 5), Sirt2.5-NES-CD (lane 6), or Sirt2.5-CD (lane 7). At 72 h posttransfection, total lysate and the cytoplasmic and nuclear fractions were prepared as described in the legend to Fig. 1. (C) HBV replication does not affect localization of Sirt2.1, Sirt2.5, and Sirt2.5-NES-CD. Huh7 cells were transfected with 3×FLAG-tagged Sirt2.1 (a to d), Sirt2.5 (i to l), or Sirt2.5-NES-CD (q to t) or cotransfected with 3×FLAG-tagged Sirt2.1 plus 1.3mer HBV WT (ayw) (e to h), Sirt2.5 plus 1.3mer HBV WT (ayw) (m to p), or Sirt2.5-NES-CD plus 1.3mer HBV WT (ayw) (u to x). Confocal images of the nuclear pore complex (NPC) taken using Alexa Fluor 647 (c, g, k, o, s, and w) and 3×FLAG-tagged Sirt2.1 (a and e), Sirt2.5 (i and m), and Sirt2.5-NES-CD (q and u) using FITC and HBc using TRITC (f, n, and v) are shown. Merged images (d, h, l, p, t, and x) are indicated. Digital images of stained cells were captured under a confocal microscope (LSM710; Zeiss, Germany). Data are representative of three independent experiments. (D) Like overexpression of Sirt2.1 or Sirt2.2, HBV replication is increased by overexpression of 3×FLAG-Sirt2.5-NES-CD. Huh7 cells were mock transfected (lane 1), transiently transfected with 4 μg of 1.3mer HBV WT (ayw) (lane 2), or cotransfected with 4 μg of 1.3mer HBV WT (awy) plus 4 μg of 3×FLAG-tagged Sirt2.1 (lane 3), Sirt2.2 (lane 4), Sirt2.5 (lane 5), Sirt2.5-NES (lane 6), Sirt2.5-NES-CD (lane 7), or Sirt2.5-CD (lane 8) construct. (E) The AKT/GSK-3β/β-catenin signaling pathway in HBV replicating cells is activated by overexpression of 3×FLAG-Sirt2.5-NES-CD (identical to overexpression of Sirt2.1 or Sirt2.2). Huh7 cells were mock transfected (lane 1), transiently transfected with 4 μg of 1.3mer HBV WT (ayw) (lane 2), or cotransfected with 4 μg of 1.3mer HBV WT (awy) plus 4 μg of 3×FLAG-tagged Sirt2.1 (lane 3), -Sirt2.2 (lane 4), Sirt2.5 (lane 5), or Sirt2.5-NES-CD (lane 6) construct. Lysates were prepared at 72 h posttransfection. The amount of transfected DNA was adjusted using pcDNA3. SDS-PAGE and immunoblotting of proteins, native agarose gel electrophoresis and immunoblotting of core particles, Southern blotting of HBV DNA, and Northern blotting of HBV RNA were performed as described in the legends to Fig. 1 and 2. Levels of acetylated α-tubulin, HBV DNA, and active AKT (pT308 and pS473) relative to those of total AKT, total β-catenin, and total/phosphorylated (pS9) GSK-3β were measured using ImageJ 1.46r. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Transfection, Staining, Microscopy, Over Expression, Construct, SDS Page, Western Blot, Agarose Gel Electrophoresis, Southern Blot, Northern Blot, Control

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Sirt2.5-AKT interaction plays no role in the HBV-associated AKT/GSK-3β/β-catenin signaling pathway. (A) Unlike the Sirt2.1-AKT interaction, the Sirt2.5-AKT interaction is weakened in HBV replicating cells. Huh7 cells were mock transfected (lanes 1, 6, and 11), transfected with 3×FLAG-Sirt2.1 (lanes 2, 7, and 12) or 3×FLAG-Sirt2.5 (lanes 4, 9, and 14), or cotransfected with 3×FLAG-Sirt2.1 plus 1.3mer HBV WT (ayw) (lanes 3, 8, and 13) or 3×FLAG-Sirt2.5 plus 1.3mer HBV WT (ayw) (lanes 5, 10, and 15). At 72 h posttransfection, total lysate and the cytoplasmic and nuclear fractions were prepared as described for Fig. 1. Total lysates and the indicated fractions were immunoprecipitated with an anti-FLAG antibody (lanes 6 to 10) or with normal IgG (lanes 11 to 15). Total lysates and the respective fractions were used as the input (lanes 1 to 5). The lysates and immunoprecipitants were subjected to SDS-PAGE and immunoblotting with anti-AKT (1:1,000; 9272S; Cell Signaling Technology), anti-FLAG M2 (1:1,000; F1804; Sigma), anti-GAPDH, and anti-H3 antibodies. (B) The Sirt2.5-associated AKT/GSK-3β/β-catenin signaling pathway is independent of HBV replication. HepG2 (lanes 1 to 6) or Huh7 (lanes 7 to 12) cells were mock transfected (lanes 1 and 7) or (co)transfected with 4 μg of 3×FLAG-Sirt2.1 (lanes 2 and 8), 4 μg of 3×FLAG-Sirt2.5 (lanes 3 and 9), 4 μg of 1.3mer HBV WT (ayw) (lanes 4 and 10), 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.1 (lanes 5 and 11), or 4 μg of 1.3mer HBV WT (ayw) plus 4 μg of 3×FLAG-Sirt2.5 (lanes 6 and 12). The amount of transfected DNA was adjusted with pcDNA3. (C) Activation of the AKT/GSK-3β/β-catenin signaling pathway by Sirt2.5 overexpression in SIRT2 KD cells does not depend on HBV replication. HepG2 cells transduced with lentiviral control shRNA (lanes 2 to 4) or Sirt2 shRNAs (shSIRT2-#2) (lanes 5 to 7) were (co)transfected with 1.3mer HBV WT (lanes 2 and 5), 1.3mer HBV WT plus 3×FLAG-Sirt2.1 (lanes 3 and 6), or 1.3mer HBV WT plus 3×FLAG-Sirt2.5 (lanes 4 and 7). Mock-transfected HepG2 cells were a negative control (lane 1). Lysates were prepared at 72 h posttransfection. SDS-PAGE and immunoblotting of proteins, native agarose gel electrophoresis and immunoblotting for core particles, and Southern blotting of HBV DNA were performed as described in the legend to Fig. 1. Levels of acetylated α-tubulin and active AKT (pT308 and pS473) relative to those of total AKT, total β-catenin, and total/phosphorylated (S9) GSK-3β were measured using ImageJ 1.46r. Data are presented as mean values from three independent experiments. Statistical significance was evaluated using Student's t test. ns, not significant; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Transfection, Immunoprecipitation, SDS Page, Western Blot, Activation Assay, Over Expression, Transduction, Control, shRNA, Negative Control, Agarose Gel Electrophoresis, Southern Blot

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Sirt2.5-mediated inhibition of HBV replication is independent of HBx. (A) The Sirt2.5-associated AKT/GSK-3β/β-catenin signaling pathway in HBV-replicating cells is not affected by the presence or absence of HBx. HepG2 cells were mock transfected (lane 1), transfected with 4 μg of 1.3mer HBV WT (ayw) (lanes 2 to 4), or transfected with 4 μg of 1.3mer HBx-deficient mutant (lanes 5 to 7) in the absence (lanes 2 and 5) or presence of either 3×FLAG-Sirt2.1 (4 μg) (lanes 3 and 6) or 3×FLAG-Sirt2.5 (4 μg) (lanes 4 and 7). (B) Sirt2.5-mediated downregulation of HBV replication is independent of HBx. HepG2 cells were mock transfected (lane 1) or cotransfected (lanes 1 to 7) as described for panel A. To supply HBx in trans, HepG2 cells were triple transfected with the 1.3-mer HBx-deficient mutant plus HA-HBx (lanes 8 to 10) plus 3×FLAG-Sirt2.1 or 3×FLAG-Sirt2.5. Lysates were prepared at 72 h posttransfection. The amount of transfected DNA was adjusted using pcDNA3 (lanes 2, 5, and 8). SDS-PAGE and immunoblotting of proteins, native agarose gel electrophoresis and immunoblotting of core particle, and Southern blotting of HBV DNA were performed as described in the legend to Fig. 1. Levels of acetylated α-tubulin and active AKT (pT308 and pS473) relative to total AKT, total β-catenin, and total and phosphorylated GSK-3β (pS9) were measured using ImageJ 1.46r. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Inhibition, Transfection, Mutagenesis, SDS Page, Western Blot, Agarose Gel Electrophoresis, Southern Blot, Control

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: HBV cccDNA-recruited Sirt2.5 associates with HKMTs such as PR-Set7 and SETDB1, which deposit epigenetic repressive markers. (A) Recruitment of HKMTs, such as PR-Set7, EZH2, SETDB1, and SUV39H1, and depositions of respective epigenetic repressive markers onto cccDNA increased in Sirt2.5-overexpressing cells. HepG2-hNTCP-C9 cells transduced with pCDH (lane 2), 3×FLAG-Sirt2.1 (lane 3), or 3×FLAG-Sirt2.5 (lane 4) were mock infected (lane 1) or infected with 1.7 × 103 GEq of HBV (lanes 2 to 4), as described in the legend to Fig. 2. Nine days after infection, chromatin solutions were prepared and subjected to immunoprecipitation with anti-FLAG, anti-Sirt2 (PA3-200 and H-95), anti-Sirt1, anti-HDAC6, anti-H3, anti-AcH3, anti-RNA Pol II, anti-H3K9me3, anti-H3K27me3, anti-H4K20me1, anti-SUV39H1, anti-PR-Set7, anti-EZH2, anti-SETDB1, and anti-SET1A antibodies or with normal rabbit polyclonal IgG (negative control). Immunoprecipitated chromatin was analyzed by PCR. Actin levels were used to ensure equal loading of lysate samples. (B) The Sirt2.5-SETDB1 interaction increased upon HBV replication. (C) The Sirt2.5–PR-Set7 interaction increased upon HBV replication. (B and C) Huh7 cells were mock transfected (lanes 1, 6, and 11), transfected with 3×FLAG-Sirt2.1 (lanes 2, 7, and 12) or 3×FLAG-Sirt2.5 (lanes 3, 8, and 13), or cotransfected with 3×FLAG-Sirt2.1 plus 1.3mer HBV WT (ayw) (lanes 4, 9, and 14) or 3×FLAG-Sirt2.5 plus 1.3mer HBV WT (ayw) (lanes 5, 10, and 15). At 72 h posttransfection, total, cytoplasmic, and nuclear fractions were prepared as described in the legend to Fig. 1. Total cell lysates and the indicated fractions were immunoprecipitated with anti-FLAG antibody (lanes 6 to 10). IgG, lysates were immunoprecipitated with normal IgG as a negative control (lanes 11 to 15). Input, total cell lysates and the respective fractions were prepared (lanes 1 to 5). The lysates and immunoprecipitants were subjected to SDS-PAGE and immunoblotting with anti-SETDB1 (B) and anti-PR-Set7 (C) antibodies. Data are representative of three independent experiments. Relative levels of immunoprecipitated SETDB1 and PR-Set7 were measured using ImageJ 1.46r. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; each relative to the control.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Transduction, Infection, Immunoprecipitation, Negative Control, Transfection, SDS Page, Western Blot, Control

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Primer sequences used for this study

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Sirt2.5 overexpression increases deposition of transcriptional repressive epigenetic markers on HBV cccDNA by repressive HKMTs. Repressive HKMTs, such as SETDB1, SUV39H1, PR-Set7, and EZH2, are recruited onto cccDNA in Sirt2.5-overexpressing cells to a greater extent than in Sirt2.1-overexpressing cells. H3K9me3 by SETDB1 and SUV39H1, H4K20me1 by PR-Set7, and H3K27me3 by EZH2 was more prominent in Sirt2.5-overexpressing cells than in Sirt2.1-overexpressing cells. Although both Sirt2.1 and Sirt2.5 interact with SETDB1 and PR-Set7 and can be recruited onto cccDNA, the Sirt2.5-SETDB1 and –PR-Set7 interactions are strengthened upon HBV replication, thereby inducing increased deposition of repressive markers in Sirt2.5-overexpressing cells. Although EZH2 and SUV39H1 cannot interact with either Sirt2.1 or Sirt2.5, more of them are recruited onto cccDNA, and deposition of repressive markers is increased in Sirt2.5-overexpressing cells. These repressive epigenetic modifications may silence cccDNA (closed state) and be transcriptionally inactive, thereby reducing viral replication.

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: Over Expression

Journal: Journal of Virology

Article Title: An Alternatively Spliced Sirtuin 2 Isoform 5 Inhibits Hepatitis B Virus Replication from cccDNA by Repressing Epigenetic Modifications Made by Histone Lysine Methyltransferases

doi: 10.1128/JVI.00926-20

Figure Lengend Snippet: Antibodies used for this study

Article Snippet: The purity of the cytoplasmic and nuclear fractions was examined by anti-GAPDH (1:5,000; sc-32233; Santa Cruz) and anti-histone H3 (1:5,000; ab1791; Abcam) antibodies, respectively. (E) Expression of Sirt2 in paired tumor and nontumor liver biopsy specimens from HBV-associated HCC patients.

Techniques: