Journal: bioRxiv

Article Title: Meteorin-like (Metrnl) adipomyokine improves glucose tolerance in type 2 diabetes via AMPK pathway

doi: 10.1101/420489

Figure Lengend Snippet: Metrnl increases GLUT4 expression by stimulating HDAC5 phosphorylation. a . Total mRNA from C2C12 cells were prepared after metrnl (100 ng/mL) treatment for the indicated times, and real-time qRT-PCR was performed using GLUT4-specific primers. For these assays, β-actin mRNA served as a positive control. b . C2C12 cells were stimulated with metrnl (100 ng/mL) for the indicate4d times. The cell lysates were analyzed by Western blotting using antibodies against GLUT4, with β-actin as a control. c . Time-dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 cells were incubated with metrnl (100 ng/mL) for the indicated times. Cell lysates were analyzed by Western blotting usingantibodies against phospho-HDAC5 (Thr 498 ), while HDAC5 served as controls. d . C2C12 cells were pre-treated with compound C (30 μM), then treated with metrnl (100 ng/mL). Cell lysates were analyzed by Western blotting using an antibody against phospho-HDAC5 (Thr 498 ); HDAC5 served as a control. e . C2C12 cells were transiently transfected with AMPKα2 siRNA or non-target siRNA. Cell lysates were analyzed by Western blotting using antibodies against phospho-HDAC5 (Thr 498 ) and AMPKα2; HDAC5 and β-actin served as controls. f . C2C12 cells were treated with metrnl (100 ng/mL). Cytosolic and nuclear proteins were extracted from the cells. The phosphorylation of HDAC5 was evaluated by Western blot analysis. HDAC5 served as a control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (α-tubulin) marker proteins. g . Representative images of phospho-HDAC5 treated with metrnl for 30?min. Scale bars, 10?μm. h . C2C12 cells were immunoprecipitated with anti-14-3-3 antibody, followed by Western blotting using anti-phospho-HDAC5. i . Representative images (phospho-HDAC5 and 14-3-3 objective images) of cells treated with metrnl for 1?h. Scale bars, 10?μm. j . The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed by qChIP analyses following 60 min of metrnl (100 ng/mL) treatment. The ChIP data represent IP values for each region’s ratio relative to the input. The results shown are from three independent experiments. * P < 0.05 and ** P < 0.01 vs. control, as indicated. Results are displayed as the mean ± SD from three experiments.

Article Snippet: HDAC5 was immunoprecipitated using an HDAC5 antibody (Novus Biologicals; Littleton, CO, USA), or an unrelated antibody (normal rabbit IgG) for a control.

Techniques: Expressing, Quantitative RT-PCR, Positive Control, Western Blot, Incubation, Transfection, Marker, Immunoprecipitation

Journal: Journal of Molecular Cell Biology

Article Title: African swine fever virus DEAD-box helicase D1133L promotes OGG1-driven incision of genomic 8-oxoG via HDAC5 deacetylation

doi: 10.1093/jmcb/mjaf029

Figure Lengend Snippet: D1133L selectively interacts with HDAC5. ( A ) Changes in Ac-D1133L levels upon treatment with TSA or NAM. HEK 293T cells were transfected with Flag-D1133L (2 μg) for 24 h and then treated with the deacetylase inhibitor TSA (2 μM) or NAM (5 mM) for 6 h, and Ac-D1133L levels were assessed and quantified. ( B ) Acetylation of endogenous D1133L in PAMs treated with TSA. PAMs were treated with TSA (2 μM) and infected with ASFV (MOI = 1) for 24 h. Ac-D1133L levels were assessed and quantified. ( C ) Interaction between D1133L and HDAC5. HEK 293T cells were transfected with Flag-D1133L (2 μg) or empty vector (EV) for 24 h and subjected to Flag IP, followed by IB using anti-HDAC antibodies. ( D ) Interaction between D1133L and HDAC5 during ASFV Infection. PAMs were infected with ASFV (MOI = 1) for 24 h and subjected to HDAC5 IP, followed by IB using anti-D1133L and anti-HDAC5 antibodies. ( E ) HDAC5 interacts with NT-D1133L. HEK 293T cells were transfected with Flag-tagged FL-D1133L, NT-D1133L, or CT-D1133L (2 μg each) or empty vector for 24 h and subjected to Flag IP, followed by IB with anti-HDAC5 antibody. Data are presented as mean ± SD ( n = 3). Statistical significance was performed using Student's t -test, compared with the control group. ** P < 0.01; * P < 0.05.

Article Snippet: Anti-HDAC5 (16166-1-AP), anti-HDAC6 (12834-1-AP), anti-NTHL-1 (11154-1-AP), anti-Myc (60003-2-Ig), anti-Flag (66008-4-Ig, 20543-1-AP), anti-HA (66006-2-Ig), anti-GAPDH (10494-1-AP), anti-β-tubulin (10094-1-AP), horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (SA00001-2), HRP-conjugated goat anti-mouse secondary antibody (SA00013-3), and 647-conjugated Myc tag monoclonal antibody were obtained from Proteintech Group.

Techniques: Transfection, Histone Deacetylase Assay, Infection, Plasmid Preparation, Control

Journal: Journal of Molecular Cell Biology

Article Title: African swine fever virus DEAD-box helicase D1133L promotes OGG1-driven incision of genomic 8-oxoG via HDAC5 deacetylation

doi: 10.1093/jmcb/mjaf029

Figure Lengend Snippet: OGG1 is involved in HDAC5-mediated deacetylation of D1133L. ( A ) Western blot analysis of OGG1 and HDAC5 levels in WT and OGG1 –/– MA104 cells. ( B ) Interaction between D1133L and HDAC5 only in WT MA104 cells. Lysates of WT and OGG1 –/– MA104 cells were transfected with Flag-D1133L (2 μg) plasmids for 24 h and subjected to Flag IP, followed by IB with anti-HDAC5 antibody. ( C ) Co-localization of D1133L, OGG1, and HDAC5. WT and OGG1 –/– MA104 cells were transfected with Flag-D1133L (2 μg) and/or Myc-HDAC5 (2 μg) for 24 h. Confocal microscopy was used to detect the co-localization by using anti-Flag, anti-OGG1, and 647-conjugated Myc tag monoclonal antibodies. DAPI was used to stain the nucleus. Scale bar, 10 μm. ( D ) Deacetylation of endogenous D1133L by HDAC5 in cells. WT and OGG1 –/– MA104 cells were transfected with Myc-HDAC5 (2 μg) for 24 h, followed by ASFV infection (MOI = 1) for an additional 24 h, and Ac-D1133L levels were assessed. ( E ) Deacetylation of transgenically expressed D1133L by HDAC5. WT and OGG1 –/– MA104 cells were transfected with Flag-D1133L (2 μg) and/or Myc-HDAC5 (2 μg) plasmids, and Ac-D1133L levels were assessed. ( F ) Deacetylation of D1133L in cells following removal of menadione treatment. WT and OGG1 –/– MA104 cells were transfected with Flag-D1133L (2 μg) plasmids for 24 h and treated with menadione (50 μM) for 2 h. Then, the medium was refreshed, and Ac-D1133L levels were assessed and quantified at the indicated time points. Data are presented as mean ± SD ( n = 3). *** P < 0.001; ** P < 0.01; * P < 0.05 ( t -test).

Article Snippet: Anti-HDAC5 (16166-1-AP), anti-HDAC6 (12834-1-AP), anti-NTHL-1 (11154-1-AP), anti-Myc (60003-2-Ig), anti-Flag (66008-4-Ig, 20543-1-AP), anti-HA (66006-2-Ig), anti-GAPDH (10494-1-AP), anti-β-tubulin (10094-1-AP), horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (SA00001-2), HRP-conjugated goat anti-mouse secondary antibody (SA00013-3), and 647-conjugated Myc tag monoclonal antibody were obtained from Proteintech Group.

Techniques: Western Blot, Transfection, Confocal Microscopy, Bioprocessing, Staining, Infection