Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

doi: 10.1073/pnas.1415589111

Figure Lengend Snippet: Fig. 2. Hyperacetylation of α-tubulin occurs despite induction of HDAC6. (A and B) HDAC6 protein levels increase concomitantly with tubulin acetylation. Acetylated tubulin and HDAC6 are both increased. **P < 0.01, n = 4–6 per group. (C) Class IIb HDAC activity is significantly increased. ***P < 0.01, n = 8–10 per group.

Article Snippet: Antibodies for immunoblotting were as follows: rabbit anti-HDAC6 antibodies were from Novus and Assay Biotech.

Techniques: Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

doi: 10.1073/pnas.1415589111

Figure Lengend Snippet: Fig. 3. Tubulin acetylation status influences aggregate formation in CryABR120G-expressing cardiomyocytes. (A) HDAC6 overexpression leads to reduced tubulin acetylation in RNCs. n = 4 per group. (B) HDAC6 overexpression increased aggregate formation in CryABR120G-infected RNCs. (Scale bar, 50 μm.) n = 50–100 cells per group. (C) HDAC6 overexpression increased levels of insoluble CryAB-positive cellular protein, confirming increased aggregate formation. *P < 0.05, n = 5 per group. (D) siRNA knockdown increased tubulin acetylation. (Scale bar, 25 μm.) (E) Aggregate formation was inhibited in CryABR120G- infected RNCs when HDAC6 was reduced, compared with cells treated with negative control (scrambled) siRNA (Negsi). n = 50–100 cells per group. (F) Re- duced HDAC6 reduced aggregate/myocyte ratios compared with CryABR120G RNCs transfected with scrambled siRNA. ***P < 0.001. (G and H) Similar effects were seen using a specific HDAC6 inhibitor, tubastatin A. ***P < 0.001. (Scale bar, 25 μm.) n = 50–100 cells per group.

Article Snippet: Antibodies for immunoblotting were as follows: rabbit anti-HDAC6 antibodies were from Novus and Assay Biotech.

Techniques: Expressing, Over Expression, Infection, Knockdown, Negative Control, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

doi: 10.1073/pnas.1415589111

Figure Lengend Snippet: Fig. 4. SAHA treatment of RNCs promotes tubulin acetylation and reduces aggregates. (A) SAHA induced α-tubulin acetylation, confirming the in- hibitory activity toward HDAC6. n = 4 per group. (B and C) SAHA treatment reduced aggregate formation in CryABR120G-infected RNCs. ***P < 0.001. (Scale bar, 50 μm.)

Article Snippet: Antibodies for immunoblotting were as follows: rabbit anti-HDAC6 antibodies were from Novus and Assay Biotech.

Techniques: Activity Assay, Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

doi: 10.1073/pnas.1415589111

Figure Lengend Snippet: Fig. 6. SAHA induces autophagy in cardiomyocytes and increased autophagy underlies the protective effects. (A) Increased autophagic vacuoles (white arrows) are increased in SAHA-treated hearts and are proximal to aggregates (*). (Scale bar, 2 μm.) (B) SAHA treatment (10 μM) in CryABR120G-infected cardiomyocytes increased autophagic flux. n = 4 per group. (C) Tubastatin A treatment (10 μM) confirms autophagy induction is mediated by HDAC6. ***P < 0.001, n = 4 per group.

Article Snippet: Antibodies for immunoblotting were as follows: rabbit anti-HDAC6 antibodies were from Novus and Assay Biotech.

Techniques: Infection

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: Pharmacologic inhibition of HDAC6 attenuates elevated vascular stiffness and protects impaired endothelial-dependent vascular relaxation in atherogenic mice. Six- to eight-week-old C57BL/6 mice were injected with adeno-associated virus (AAV) encoding a PCSK9 gain-of-function mutant and then were fed a high-fat diet (HFD) for 18 weeks in the presence or absence of the HDAC6 inhibitor tubacin (0.5 mg/kg daily). (A) Pulse wave velocity (PWV) and (B) body weight was measured every 2 weeks until the end of the diet regimen (18 weeks). ∗ p < 0.05 vs. vehicle group; n = 10–12. Vascular reactivity in response to (C) acetylcholine and (D) sodium nitroprusside (SNP) was measured by wire myography of isolated aortic rings. ∗ p < 0.05 vs. control group; n = 6–8 mice. (E) Oil Red O staining was used to determine plaque burden in isolated aortas. (F) Quantification of plaque area presented in (E) . ∗∗ p < 0.05 vs. normal diet group; # p < 0.05 vs. PCSK9aav + HFD group; n = 3. (G) Frozen aortic root cross sections were stained with Oil Red O. The graph in the lower right quadrant shows quantification of plaque area. ∗ p < 0.05 vs. normal diet group; # p < 0.05 vs. PCSK9aav + HFD group; n = 3.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Inhibition, Injection, Virus, Mutagenesis, Isolation, Control, Staining

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: Pharmacologic inhibition of NEDDylation pathway regulates endothelial HDAC6 activity and modulates α-tubulin organization. (A) Human aortic endothelial cells (HAECs) were exposed to either DMSO or MLN4924 (1 μM) for 18 h, and cell lysates were subjected to western blotting with antibodies to acetylated α-tubulin, HDAC6, or total α-tubulin. (B) Similar experiments were performed with two doses of MLN4924 (0.1 and 1 μM); n = 3. (C) Densitometric analysis of HDAC6 and acetylated α-tubulin levels from panels (A,B) . ∗ p < 0.05 vs. vehicle group; # p < 0.05 vs. MLN4925 (0.1 μM) group; n = 3. (D) HEK293 cells were transfected with empty vector (pcdna3.1) or FLAG-tagged HDAC6. After 24 h, MLN4924 or DMSO (control) was added to the media and cultured for another 18 h. Cell lysates were immunoblotted (IB) with acetylated α-tubulin and FLAG antibodies. (E) Left: A confluent monolayer of HAECs was exposed to DMSO or MLN4924 (1 μM) for 18 h. Cells were immunofluorescently stained for HDAC6 (green), α-tubulin (red), and DAPI (blue). Right: Quantification of gap area between adjacent cells normalized to the number of nuclei. ∗ p < 0.05 vs. control group; n = 3.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Inhibition, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Control, Cell Culture, Staining

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: MLN4924 fails to increase acetylated tubulin in tubacin-pretreated HAECs. (A) HAECs were pretreated with tubacin for 4 h before addition of MLN4924 (1 μM). After an 18 h incubation, cells were immunoblotted (IB) with antibodies to acetylated α-tubulin, HDAC6, and total α-tubulin. (B,C) Densitometric analysis of HDAC6 and acetylated α-tubulin levels presented in (A) . ∗ p < 0.05 vs. DMSO (control) group; n = 3.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Incubation, Control

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: NEDD8 binds HDAC6 within the ubiquitin binding BUZ domain. (A) HEK293 cells expressing FLAG-tagged full-length HDAC6 (FLAGHDAC6WT), HA-NEDD8, and UBC12 were immunoprecipitated (IP) with either isotype control IgG or HA antibodies. The purified conjugates were immunoblotted (IB) with FLAG, HA, or UBC12 antibodies. (B) HEK293 cells expressing FLAG-tagged full-length HDAC6 (FLAGHDAC6WT) or ubiquitin binding domain truncated HDAC6 mutant (FLAGHDAC6ΔBUZ) together with HA-NEDD8 and UBC12 were immunoprecipitated with either isotype control IgG or HA antibodies. The purified conjugates were immunoblotted with FLAG, HA, or UBC12 antibodies. (C) Densitometric analysis of NEDDylated FLAGHDAC6 WT and FLAGHDAC6ΔBUZ normalized to total HDAC6. ∗∗ p < 0.05 vs. FLAGHDAC6 WT group; n = 3.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Ubiquitin Proteomics, Binding Assay, Expressing, Immunoprecipitation, Control, Purification, Mutagenesis

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: Pharmacologic inhibition of NEDDylation pathway blocks OxLDL-induced HDAC6 activity. (A) HAECs pretreated with MLN4924 (1 μM) for 2 h were exposed to either phosphate-buffered saline (control) or increasing concentrations of OxLDL (50, 100 μg/mL) for 6 h. Cell lysates were immunoblotted (IB) with acetylated α-tubulin and total α-tubulin antibodies. (B) Densitometric analysis of acetylated α-tubulin. ∗ p < 0.05 vs. control group; # p < 0.05 vs. OxLDL group; n = 3.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Inhibition, Activity Assay, Saline, Control

Journal: Frontiers in Physiology

Article Title: Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo : A Role for HDAC6 Neddylation

doi: 10.3389/fphys.2021.675724

Figure Lengend Snippet: Schematic of proposed mechanism for the role of HDAC6 in endothelial dysfunction and atherogenesis. OxLDL stimulates HDAC6 activity by enhancing HDAC6 NEDDylation. This leads to suppression of the endothelial enzymes NOS and CSEγ, thereby reducing NO and H 2 S. These events result in EC dysfunction and atherosclerosis.

Article Snippet: The catalytically inactive mutant of HDAC6 (H216/611A, Addgene plasmid #30483) and C-terminal zinc finger domain (binder of ubiquitin zinc finger, BUZ Addgene plasmid #30484) deleted mutant were kind gifts from Tso-Pang Yao ( ).

Techniques: Activity Assay

Journal: Kidney360

Article Title: Dynein-Mediated Trafficking: A New Mechanism of Diabetic Podocytopathy

doi: 10.34067/KID.0006852022

Figure Lengend Snippet: Antibodies and fluorescent probes (immunofluorescent, Western blotting, immunoprecipitation]

Article Snippet: Rabbit anti-HDAC6 , WB: 1:1000 , Novus , NBP1-78981.

Techniques: Western Blot, Immunoprecipitation