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

Article Title: Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6

doi: 10.1073/pnas.0908908106

Figure Lengend Snippet: Sulforaphane treatment inhibits HDAC6. Tubulin dimers were either incubated without recombinant HDAC6 or with recombinant HDAC6 in the presence of vehicle, sulforaphane, or TSA. Levels of HDAC6, acetylated tubulin, and tubulin by Western blot were quantified.

Article Snippet: Two micrograms of recombinant HDAC6 (Biomol) were used in a cell-free tubulin deacetylase assay with 25 μg of MAP-rich polymerized tubules (Cytoskeleton), similar to Hubbert et al. ( 14 ).

Techniques: Incubation, Recombinant, Western Blot

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

Article Title: Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6

doi: 10.1073/pnas.0908908106

Figure Lengend Snippet: Ectopic overexpression of HDAC6 attenuates sulforaphane-mediated AR and HDAC6 protein depletion, and HDAC6 siRNA recapitulates the findings seen with sulforaphane. (A) LNCaP cells were transfected with pCDNA3.1 or FLAG-HDAC6. Cells were then treated with either vehicle or 15 μM sulforaphane. Intensity values for bands by Western blot in the respective vehicle controls were set to 1. (B) LNCaP cells were transfected with siRNA to either the luciferase gene (si LUC) or HDAC6 gene (si HDAC6). Levels of protein expression by Western blot were quantified, and the bands from the HDAC6 siRNA samples were compared to the luciferase control samples from the same time point.

Article Snippet: Two micrograms of recombinant HDAC6 (Biomol) were used in a cell-free tubulin deacetylase assay with 25 μg of MAP-rich polymerized tubules (Cytoskeleton), similar to Hubbert et al. ( 14 ).

Techniques: Over Expression, Transfection, Western Blot, Luciferase, Expressing, Control

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

Article Title: Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6

doi: 10.1073/pnas.0908908106

Figure Lengend Snippet: Model of sulforaphane attenuation of AR signaling via HDAC6 inactivation. Normally, HSP90 is deacetylated by HDAC6, which enables it to chaperone client proteins such as AR. HDAC6 also deacetylates alpha-tubulin. With sulforaphane treatment, HDAC6 is inhibited or targeted for protein degradation. This leads to hyperacetylated alpha-tubulin; the functional sigificance of this remains unclear. The HDAC6 inactivation with sulforaphane also leads to hyperacetylated, inactive HSP90 protein, which dissociates from AR, and AR is then targeted for protein degradation. Consequently, AR binding to its target gene androgen response elements (ARE), including TMPRSS2-ERG, is diminished, which reduces their expression.

Article Snippet: Two micrograms of recombinant HDAC6 (Biomol) were used in a cell-free tubulin deacetylase assay with 25 μg of MAP-rich polymerized tubules (Cytoskeleton), similar to Hubbert et al. ( 14 ).

Techniques: Functional Assay, Binding Assay, Expressing

Journal: Animal Cells and Systems

Article Title: Histone deacetylase 6 interference protects mice against experimental stroke-induced brain injury via activating Nrf2/HO-1 pathway

doi: 10.1080/19768354.2019.1601132

Figure Lengend Snippet: HDAC6 was up-regulated in brain I/RI mice. (A) RT-qPCR was used to analyze mRNA expression levels of HDAC6. ## P < 0.01 vs. NC group. (B) Western blot was used to analyze the protein levels of HDAC6 in brain I/R-injured MCAO group and the shRNA-HDAC6 lentivirus transfected MCAO mice (sham + shHDAC6 group). ** P < 0.01 vs. sham group, ## P < 0.01 vs. MCAO group. (C) RT-qPCR was used to analyze mRNA expression levels of HDAC6. ** P < 0.01 vs. sham group, ## P < 0.01 vs. MCAO group.

Article Snippet: The recombinant shRNA-HDAC6 and scramble shRNA lentivirus plasmid were obtained from Genepharma (Shanghai, China), and the virus titer was 1 × 10 9 UT/mL.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, shRNA, Transfection

Journal: Animal Cells and Systems

Article Title: Histone deacetylase 6 interference protects mice against experimental stroke-induced brain injury via activating Nrf2/HO-1 pathway

doi: 10.1080/19768354.2019.1601132

Figure Lengend Snippet: HDAC6 exacerbated brain damage and oxidative stress in brain I/RI mice. (A and C) Brain TTC staining was used to measure the cerebral infarct areas. (B) The neurological scores were compared. (D–F) The protein levels of oxidative stress-related factors including 3-NT, 4-HNE and 8-OHdG in serum were measured by ELISA. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. MCAO group.

Article Snippet: The recombinant shRNA-HDAC6 and scramble shRNA lentivirus plasmid were obtained from Genepharma (Shanghai, China), and the virus titer was 1 × 10 9 UT/mL.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Animal Cells and Systems

Article Title: Histone deacetylase 6 interference protects mice against experimental stroke-induced brain injury via activating Nrf2/HO-1 pathway

doi: 10.1080/19768354.2019.1601132

Figure Lengend Snippet: HDAC6 inhibited Nrf2/HO-1 pathway in brain I/RI mice. (A–D) The protein levels of Nrf2 and HO-1 in cytoplasm, and Nrf2 in cytoblast were determined by Western blot. ** P < 0.01 vs. sham group, ## P < 0.01 vs. MCAO group.

Article Snippet: The recombinant shRNA-HDAC6 and scramble shRNA lentivirus plasmid were obtained from Genepharma (Shanghai, China), and the virus titer was 1 × 10 9 UT/mL.

Techniques: Western Blot

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 1. SARS-CoV-2 3CLpro is released from infected cells by cell lysis and secreted by an unconventional protein-secretion mechanism (A) Immunoblots of 3CLpro precipitated by 33% trichloroacetic acid (TCA) from the conditioned media or in lysates from mock-infected or A549-ACE2 cells 48 hpi with SARS-CoV-2 (MOI 4, n = 5, N = 2). stds, 3CLpro standards. Densitometry of 3CLpro relative to 10 ng 3CLpro standard. (B) 3CLpro, b-tubulin, and GAPDH immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media or cell lysate from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi with MOI 0.1 or 1.0. Dotted line, intracellular 3CLpro/tubulin ratio in MOI 1.0 cell lysate (n = 3/group). (C) Immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media compared to matched lysates from SARS-CoV-2-infected Vero76 cells 48 hpi (MOI 1) or mock-infected cells (n = 3/group). 3CLpro ppt, 10 ng of 3CLpro precipitated from cell-naive media as a control; std, 10 ng of 3CLpro standard. (D) Immunoblots of 3CLpro in conditioned media concentrated by ultrafiltration and cell lysates from HEK293 cells transfected with 3CLpro expression plasmid for 48 h (n = 8, N = 2). (E) LDH activity in conditioned medium collected from 3CLpro transfected HEK293 cells (48 h) compared with the maximum amount of LDH released from lysed HEK293 cells (n = 6, N = 2). Absorbance was measured at 490 nm with 680 nm correction. (F) Immunoblots and densitometry of 3CLpro in concentrated conditioned medium and cell lysates from HEK293 cells transiently transfected with plasmids encoding 3CLpro or catalytic inactive 3CLpro Cys145Ala for 48 h (n = 5, N = 2). (G) Immunoblots and densitometry of 3CLpro or IFN-l1 after 24 h culture ± Golgi inhibitor 2 mM monensin (n = 6, N = 2). (H) 3CLpro, GSDMD, and b-actin loading control immunoblots and GSDMD densitometry of lysates from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi, MOI 0.1 or 1.0 (n = 3/group). (I) Schematic of candidate mechanisms of 3CLpro release from SARS-CoV-2-infected cells. Antibodies: anti-3CLpro (1:1,000, in-house), anti-b-tubulin (1:2,000, clone: BT7R), anti-b-actin (1:2,000, ab8226), anti-GAPDH (1:2,000, clone: GA1R), anti- GSDMD N-terminal antibody (1:500, clone: W18029B), and anti-FLAG M2 (IFN-l1 in G) (1:1,000, F3165). Data are shown as mean ± SD; statistical analysis of two groups was performed by two-tailed unpaired t test, three or more groups by one-way ANOVA with Tukey’s post hoc test, with a significance threshold of p < 0.05.

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Infection, Lysis, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 2. SARS-CoV-2 3CLpro cleaves GSDMD at LQ29Y30SS to block GSDMD pore formation, whereas caspase cleavage generates func- tional GSDMD pores that directly secrete 3CLpro and nucleocapsid protein (A) Schematic of 3CLpro cleavage sites (non-prime side [P] in blue, prime side [P0] in red) and the caspase cleavage site (violet) in human GSDMD. Y, scissile bond.

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Blocking Assay

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 3. SARS-CoV-2 3CLpro is secreted through caspase-activated GSDMD and GSDME pores and retains activity in serum to dampen platelet activation (A) Recombinant 3CLpro (2 mM) was incubated with or without recombinant GSDME (3 mg) for 2 h at 37C and resolved on 4%–12% reducing SDS-PAGE stained with Coomassie (N = 2). 3CLpro with C-terminal 33FLAG-Myc-63His tag was used. (legend continued on next page)

Article Snippet: Full-length human GSDMD 1–484 expression construct with C-terminal FLAG tag was from Genscript (clone ID: OHu03821, accession # GenBank: NM_024736.7), which was used to generate 3CLpro- or caspase-cleaved analogues: GSDMD 1–270 (Addgene plasmid #218229), GSDMD 1–275 (Addgene plasmid #218230), GSDMD 30–484 (Addgene plasmid #218231), and GSDMD 30–270 (Addgene plasmid #218232).

Techniques: Activity Assay, Activation Assay, Recombinant, Incubation, SDS Page, Staining

Journal: Cell Reports

Article Title: Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

doi: 10.1016/j.celrep.2022.110736

Figure Lengend Snippet: DARPin F10 inhibits ZnF-Ub interaction in vitro and in cells (A) Schematic of HDAC6, showing the catalytic domains (CD1, CD2) and the zinc finger domain (ZnF, amino acid 1,108–1,215) used to select binders. Generic nanobody and DARPin structures (PDB: 1I3V and PDB: 2QYJ ) are shown. The HDAC6 sequence (aa 1,153–1,190) at top shows the Ub binding motifs (framed red) (Uniprot: Q9UBN7 ) ( Ouyang et al., 2012 ). (B) F10 blocks ZnF-Ub interaction in vitro . Purified His-tagged ZnF (aa 1,108–1,215), Flag A10 or F10 and mono-Ub were mixed; following incubation, DARPins and associated proteins were pulled down with anti-Flag beads. The precipitate was analyzed by immunoblotting, using antibodies against His, Ub, and Flag. PD, pull down; FT, flow through. (C) In 293T cells, ZnF-Ub interaction is disrupted by F10, as monitored by a split-GFP assay. ZnF (aa 1,108–1,215) and Ub were fused to separate GFP fragments so that ZnF-Ub interaction is required to reconstitute a functional GFP molecule (scheme at top). GFP beta strands are GFP-(1–9), GFP-10, and GFP-11. A mutant ZnF (W1182A, ZnF m ) was used as a control. An mRuby plasmid was included in all transfections (red signal). Scale bar: 1 mm. (D) Immunoprecipitation of endogenous HDAC6 by F10. A GFP fusion protein to F10 or to control E3_5 was transiently expressed in A549 cells, and the DARPins were immunoprecipitated with GFP-trap beads. The immunoprecipitate (IP) was analyzed by immunoblotting, using antibodies against GFP or HDAC6. (E) Mass spectrometry analysis to determine the F10 interactome. Immunoprecipitate (D above) was analyzed by mass spectrometry, and enriched proteins are annotated.

Article Snippet: Human monoclonal HDAC6 antibody , Cell Signaling , Cat#7558; RRID: AB_10891804.

Techniques: In Vitro, Sequencing, Binding Assay, Purification, Incubation, Western Blot, Split GFP Assay, Functional Assay, Mutagenesis, Control, Plasmid Preparation, Transfection, Immunoprecipitation, Mass Spectrometry

Journal: Cell Reports

Article Title: Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

doi: 10.1016/j.celrep.2022.110736

Figure Lengend Snippet: 2.55-Å crystal structure shows the detail of DARPin F10 inhibition on Ub recruitment (A) F10 bound to HDAC6 ZnF. F10, ZnF (aa 1,108–1,215), and Zn 2+ ions are colored purple, green, and gray. F10 α helices (α1 to α10) and β turns (L1 to L4) are numbered starting from the N-terminus (PDB: 7ATT ). (B) ZnF conformation alignment in two binding forms. Green shows the ZnF structure bound to F10, and cyan shows the ZnF bound to Ub (PDB: 3GV4 ). Right: zoomed-in view of the Ub binding “gatekeeper” amino acids, Arg1155 and Tyr1156. The shifting of side chains is indicated by dashed lines with the distance labeled. (C) Zoomed-in views of the ZnF-Ub-binding pocket. The ZnF-F10 structure is superimposed onto the ZnF-Ub C-terminal tail structure (PDB: 3GV4 ). The Ub C-terminal tail is shown in surface representation (yellow): F10 L3 backbone clashes with the Ub tail. The region from His101 to Gly103 is indicated by the dashed line rectangle (right). (D) Affinities of ZnF for F10 or free Ub by ITC assays. ZnF-F10: ΔS = 20.6 cal/mol/deg, N = 1.01. ZnF-Ub: ΔS = −12.8 cal/mol/deg, N = 1.46.

Article Snippet: Human monoclonal HDAC6 antibody , Cell Signaling , Cat#7558; RRID: AB_10891804.

Techniques: Inhibition, Binding Assay, Labeling

Journal: Cell Reports

Article Title: Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

doi: 10.1016/j.celrep.2022.110736

Figure Lengend Snippet: DARPin F10 interferes with IAV infection (A) F10 can be efficiently degraded. Immunoblotting with lysates of F10-FKBP cells treated for the indicated times with dTAG (2 μM). The leftmost lane shows the parental A549 cells. The membrane was probed with antibodies against HDAC6, α-tubulin, and F10 (HA-F10-FKBP F36V , detected with anti-HA). (B) F10 impairs IAV infection. A549 WT, F10-FKBP, or FKBP cells were infected with IAV at a MOI of 0.05 (n = 3), and culture supernatants were collected every 12 h until 72 h. Viral titer was quantified by plaque assay. Statistical analysis was done with one-way ANOVA test; p values show the significant (< 0.05) difference between cell lines expressing or not F10. Data are represented as mean ± SD. (C) Treatment with dTAG restores IAV susceptibility. The indicated cell lines (−/+ dTAG pre-treatment) were infected with IAV at an MOI of 0.05 (n = 3). Culture supernatants were analyzed as in (B); p values show the significant (< 0.05) difference between A549 WT and F10-FKBP cells. Data are represented as mean ± SD. (D) Effect of F10 on a single IAV life cycle. The indicated cell lines were infected with IAV at an MOI of 10 (n = 3), and culture supernatants were collected every 2 h up to 8 h. Viral titer was analyzed as in (C) above. (E) IAV uncoating is impaired by F10. Left panels: confocal microscopy visualization of uncoating, staining for the capsid M1 protein (green). Parental A549 or F10-FKBP cells (−/+ dTAG pre-treatment) were used for IAV infection, and M1 localization was analyzed 3.5 hpi. Bafilomycin A1 treatment was used to block endosomal acidification. Total protein was stained to visualize the cell body (red); scale bar, 20 μm. Right panel, quantification of the image analysis in the different samples (n = 3); ca. 30 cells were selected per view (six to nine views for each condition), and M1 fluorescence intensity was analyzed. Statistical analysis as in (B); p values show the significant (< 0.05) difference against untreated A549 WT cells. Data are represented as mean ± SD.

Article Snippet: Human monoclonal HDAC6 antibody , Cell Signaling , Cat#7558; RRID: AB_10891804.

Techniques: Infection, Western Blot, Membrane, Plaque Assay, Expressing, Confocal Microscopy, Staining, Blocking Assay, Fluorescence

Journal: Cell Reports

Article Title: Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

doi: 10.1016/j.celrep.2022.110736

Figure Lengend Snippet: DARPin F10 impairs cellular granules formation (A) Aggresome formation was induced with MG132 in indicated cells (−/+ dTAG pre-treatment). Aggresomes were detected by staining for HDAC6 and Ub; DAPI was used to stain the nucleus. Scale bar, 20 μm. (B) Quantification of aggresome formation by Ub signal (n = 3). The graph shows the percentage of aggresome-positive cells; each data point represents the percentage obtained in a randomly chosen micrograph containing ca. 50 cells. Statistical analysis was done by one-way ANOVA; p values refer to significant (< 0.05) differences between samples. Data are represented as mean ± SD. (C) SGs were induced with NaAsO 2 in indicated cells (−/+ dTAG pre-treatment); SGs were visualized and quantified by staining for G3BP1. Representative pictures are shown. Scale bar, 20 μm. (D) Quantification of the number of SGs per cell area. Each data point represents the number of SGs per cell area in a sample containing one or a few (ca. 2 to 5) cells. Data were collected in five independent experiments (n = 5). Statistical analysis as in (B).

Article Snippet: Human monoclonal HDAC6 antibody , Cell Signaling , Cat#7558; RRID: AB_10891804.

Techniques: Staining

Journal: Cell Reports

Article Title: Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways

doi: 10.1016/j.celrep.2022.110736

Figure Lengend Snippet:

Article Snippet: Human monoclonal HDAC6 antibody , Cell Signaling , Cat#7558; RRID: AB_10891804.

Techniques: Produced, Recombinant, Virus, Ubiquitin Proteomics, Mutagenesis, SYBR Green Assay, Reverse Transcription, RNA Sequencing, Transfection, Plasmid Preparation, Software, Microscopy

Journal: Cell

Article Title: G3BP1 is a tunable switch that triggers phase separation to assemble stress granules

doi: 10.1016/j.cell.2020.03.046

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-HDAC6 antibody , Abcam , ab133493.

Techniques: Recombinant, Protease Inhibitor, Electron Microscopy, Lysis, Staining, Isolation, Mutagenesis, Sequencing, Plasmid Preparation, Software, Imaging

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: HDAC6 is involved in DSB repair. ( A ) pEJ5-GFP U2OS or DR-U2OS cells were transfected with HDAC6 or negative control (NC) siRNAs and subjected to NHEJ and HR assays, separately. ( B ) pEJ5-GFP U2OS or DR-U2OS cells were transfected with Myc-HDAC6 or empty vector and subjected to NHEJ and HR assays, separately. (C, D) HeLa cells were transfected with HDAC6 or NC siRNAs for 48 h, treated with 10 μM VP16 for 2 h, and washed three times with fresh medium before recovery for the indicated times. All samples were then subjected to a comet assay. Representative images ( C ) and statistical analyses ( D ) are shown. (E, F) HeLa cells were transfected with Flag-HDAC6 plasmids for 48 h. After 3 Gy irradiation, the cells were counted and seeded for the colony formation assays. For the control (Ctr) group, 500 cells were seeded in each plate. For the IR-treated group, 5000 cells were seeded in each plate. Representative images ( E ) and statistical analyses ( F ) are shown. (G, H) HDAC6 WT or HDAC6 KO HCT116 cells underwent 3 Gy irradiation (IR) and were counted and seeded for colony formation assays. For the control (Ctr) group, 500 cells were seeded in each plate. For the IR-treated group, 5000 cells were seeded in each plate. Representative images ( G ) and statistical analyses ( H ) are shown. All data represent the means ± SD ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Transfection, Negative Control, Plasmid Preparation, Single Cell Gel Electrophoresis, Irradiation, Control

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: HDAC6 interacts with H2A/H2A.X in response to DSBs, inhibiting H2A/H2A.X ubiquitination. ( A ) HeLa cells were transfected with Flag-HDAC6 or an empty plasmid for 48 h. Chromatin fractions were subjected to immunoprecipitation with an anti-Flag antibody. Western blots of indicated proteins are shown. ( B ) Whole cell lysates from HeLa cells were extracted and immunoprecipitated using an anti-HDAC6 (up) or an anti-H2A.X (down) antibody. Rabbit IgG was used as a negative control. Western blotting was performed with the indicated antibodies. ( C ) GST or GST-H2A.X fusion proteins were expressed in bacteria, purified, and then incubated with His-HDAC6 protein. Western blotting was performed to detect HDAC6 protein levels and Coomassie Brilliant Blue (CBB) staining was performed to detect GST or GST-H2A.X levels. The asterisk indicates the corresponding protein bands. ( D ) HCT116 cells were co-transfected with Flag-H2A.X, Myc-HDAC6, or an empty plasmid for 48 h. Flag-H2A.X was immunoprecipitated from whole cell lysates and eluted for western blotting with the indicated antibodies (FK2: anti-ubiquitin conjugate antibody, Pan-me: anti-pan methyl Lysine antibody, p-S/T: anti-Phospho-(Ser/Thr) antibody, Pan-ac: anti-acetyl lysine antibody) to detect post-translational modifications. ( E ) HDAC6 WT or HDAC6 KO HCT116 cells were transfected with Flag-H2A.X and HA-ub, with or without Myc-HDAC6 for 48 h, before they were exposed to IR (10 Gy) and released for 1 h. Flag-H2A.X proteins were immunoprecipitated from the whole cell lysates and eluted for western blotting to detect changes in ubiquitination status.

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Negative Control, Bacteria, Purification, Incubation, Staining

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: HDAC6 is associated with the H2A/H2A.X ubiquitination signaling cascade in DSB repair. (A–E) All cells were irradiated with 10 Gy followed by release for 1 h, after transfection with the indicated plasmids or siRNAs. ( A ) HeLa cells were transfected with Myc-HDAC6 or an empty plasmid for 48 h. Whole cell lysate and chromatin fractions were subjected to western blotting. ( B ) HeLa cells were transfected with HDAC6 or negative control (NC) siRNAs for 48 h. Whole cell lysate and chromatin fractions were subjected to western blotting. ( C ) HDAC6 KO HCT116 cells were transfected with Myc-HDAC6 or empty plasmid for 48 h. Whole cell lysate and chromatin fractions were subjected to immunoblotting. ( D, E ) HeLa cells were transfected with HDAC6 or negative control (NC) siRNAs for 48 h. The cells were then fixed, and the samples were labeled with the indicated antibodies. Representative images (D) and statistical analyses (E) are shown. The data represent the means ± SD ( n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Ubiquitin Proteomics, Irradiation, Transfection, Plasmid Preparation, Western Blot, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: HDAC6 regulates the H2A/H2A.X ubiquitination signaling cascade in an RNF168-dependent manner during DSB repair. ( A ) HeLa cells were transfected with Flag-HDAC6 or an empty plasmid for 48 h. Whole cell lysates were subjected to immunoprecipitation with an anti-Flag antibody before western blotting. ( B ) HeLa cells were transfected with Myc-HDAC6 or empty plasmid for 48 h and exposed to 10 Gy irradiation (IR) and released for 1 h. Chromatin fractions were subjected to immunoprecipitation with an anti-H2A.X antibody followed by western blotting. Rabbit IgG was used as a negative control. (C, D) HeLa cells were co-transfected with GFP-RNF168 and mCherry-HDAC6 or an empty plasmid for 48 h and then subjected to laser micro-IR. Images were captured every 10 s for 200 s ( C ), and the IR path signal intensity was quantified ( D ). ( E ) Recombinant His-HDAC6 and GST-RNF168 were subjected to in vitro ubiquitination assays in the presence of ATP, E1 (UBE1), E2 (UbcH5c), ub (ubiquitin) and H2A/H2B dimer as indicated. Western blotting was performed with the indicated antibodies. (F, G) HeLa cells were transfected with the indicated plasmids for 48 h and then exposed to 10 Gy irradiation and released for 1 h. Whole cell lysate and chromatin fractions were analyzed by western blotting with the indicated antibodies. WT: wild-type; ΔDC1: DAC1 domain deleted; ΔDC2: DAC2 domain deleted; ΔZnF: ZnF-UBP domain deleted ( F ); HDAC6 catalytic mutant (Y386F/Y782F) ( G ). ( H ) HeLa cells were co-transfected with Flag-H2A.X, HA-ub, with Myc-HDAC6 WT or Myc-HDAC6 2YF (catalytic mutant) for 48 h and then exposed to 10 Gy irradiation and released for 1 h. The Flag-H2A.X proteins were immunoprecipitated from the whole cell lysates and eluted to detect the ubiquitination changes by western blotting with the indicated antibodies. All data represent the means ± SD.

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Irradiation, Negative Control, Recombinant, In Vitro, Mutagenesis

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: Nuclear HDAC6 is displaced from chromatin and degraded via the proteasome in response to DSBs. ( A , B ) HeLa cells were exposed to an increasing dose of irradiation (IR) and released after 1 h or treated with 10 μM etoposide (VP16) for the indicated time. Then, whole cell lysate (β-actin), nuclear (Lamin-B1), and chromatin (H3) fractions were isolated and analyzed by western blotting. ( C ) HeLa cells were treated with 10 μM MG132 for 3 h or 50 μM CHQ for 12 h, then exposed to 10 Gy IR and released for 1 h before analysis of the whole cell lysate and nuclear fractions by western blotting. (D–F) HeLa cells were co-transfected with Flag-HDAC6 and HA-ub ( D ), Flag-HDAC6 and different HA-ubiquitin constructs (WT: wild type ubiquitin, K48R: K48-mutant ubiquitin, K63R: K63-mutant ubiquitin) ( E ) or Flag-HDAC6 wild-type (WT) or mutant (K116R) ( F ) for 48 h. Then, the cells were treated with 10 μM MG132 for 2 h, exposed to 10 Gy IR, and released for 1 h. The nuclear fractions were then subjected to immunoprecipitation with an anti-Flag antibody and analyzed by western blotting.

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Irradiation, Isolation, Western Blot, Transfection, Ubiquitin Proteomics, Construct, Mutagenesis, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: RNF168 directly interacts with HDAC6 and mediates DSB-induced nuclear HDAC6 degradation. ( A ) HeLa cells were transfected with Flag-HDAC6 or an empty plasmid for 48 h. Whole cell lysates were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by western blotting. ( B ) Whole cell lysates from HeLa cells were extracted and subjected to immunoprecipitation using an anti-RNF168 (up) or an anti-HDAC6 (down) antibody and subsequent analysis by western blotting. Rabbit IgG served as a negative control. ( C ) HeLa cells were transfected with Flag-HDAC6 or an empty plasmid for 48 h. Then, the cells were treated with 10 μM MG132 for 2 h, exposed to 10 Gy irradiation (IR), and released for the indicated times. The nuclear fractions were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by western blotting. ( D ) GST or GST-RNF168 fusion proteins were incubated with His-HDAC6 protein. Western blotting was performed to detect HDAC6 protein levels, and Coomassie Brilliant Blue (CBB) staining was performed to detect GST or GST-RNF168 levels (indicated by the asterisk). ( E ) HeLa cells were transfected with RNF168 or negative control (NC) siRNAs for 48 h then exposed to 10 Gy IR and released for 1 h before the whole cell lysate and nuclear fractions were analyzed by western blotting. ( F ) RNF168-WT or RNF168 KO HCT116 cells were transfected with Flag-RNF168 or empty plasmids for 48 h, then exposed to 10 Gy IR and released after 1 h before the whole cell lysate and nuclear fractions were analyzed by western blotting. ( G ) HCT116 cells were transfected with RNF168 or negative control (NC) siRNAs for 12 h, then co-transfected with the indicated plasmids for 48 h. Then, the cells were treated with 10 μM MG132 for 2 h and exposed to 10 Gy IR and released for 1 h before the nuclear fractions were subjected to immunoprecipitation with an anti-Flag antibody and analyzed by western blotting. ( H ) Recombinant His-HDAC6 and GST-RNF168 were subjected to in vitro ubiquitination assays performed in a reactive system containing E1 (UBE1), E2 (UBE2D3), HDAC6 (substrate), and ubiquitin with increasing RNF168 (E3 ligase). The reactions were analyzed by western blotting. ( I ) HDAC6 KO cells were transfected with Flag-HDAC6 wild-type (WT) or mutant (K116R) for 48 h, then exposed to 10 Gy IR and released after 1 h before the whole cell lysate and chromatin fractions were analyzed by western blotting.

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Negative Control, Irradiation, Incubation, Staining, Recombinant, In Vitro, Ubiquitin Proteomics, Mutagenesis

Journal: Nucleic Acids Research

Article Title: The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair

doi: 10.1093/nar/gkad631

Figure Lengend Snippet: RNF168-mediated nuclear HDAC6 degradation is beneficial for DSB repair and cell survival. (A, B) pEJ5-GFP U2OS ( A ) and DR-U2OS ( B ) cells were transfected with Flag-HDAC6 wild-type (WT) or mutants (K116R or 2YF) for 48 h and subjected to NHEJ (A) and HR (B) assays, respectively. (C, D) HeLa cells were transfected with Flag-HDAC6 wild-type (WT) or mutants (K116R or 2YF) for 48 h, exposed to 10 Gy irradiation (IR), and then allowed to recover under normal conditions for the indicated times. All samples were then analyzed by comet assay. Representative images ( C ) and data analysis ( D ) are shown. (E, F) HDAC6-WT, HDAC6 KO, HDAC6 KO re-transfected Flag-HDAC6(WT), and Flag-HDAC6(K116R) stably expressing cells were exposed to 3 Gy IR and released for 8 h. The cells were then treated with colcemid (0.03 μg/ml) for an additional 6 h before chromosomal abnormality analysis. Representative images ( E ) and data analysis ( F ) are shown. (G, H) HeLa cells were transfected with Flag-HDAC6 wild-type (WT) or mutants (K116R or 2YF) for 48 h and then exposed to 3 Gy IR. The cells were counted and seeded for colony formation assays. For the control (Ctr) group, 500 cells were seeded in each plate, while for the IR-treated group, 5000 cells were seeded in each plate. ( I, J ) HDAC6-WT, HDAC6 KO, HDAC6 KO re-transfected Flag-HDAC6 (WT), and HDAC6 KO re-transfected Flag-HDAC6 (K116R) stably expressing cells were exposed to 3 Gy IR before being counted and seeded for colony formation assays. For the control (Ctr) group, 500 cells were seeded in each plate, while for the IR-treated group, 5000 cells were seeded in each plate. (K ) A model of the role played by HDAC6 in DSB repair. All data represent the means ± SD ( n = 60 for E–F, I–J; n = 3 for A–D, G–H; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Various fragments of HDAC6 (DAC1 domain, 1–440 aa; DAC2 domain, 441–835 aa; ZnF domain, 836–1215 aa) were cloned into the p3 × FLAG-CMV10 or pET28a vector (Addgene, USA).

Techniques: Transfection, Irradiation, Single Cell Gel Electrophoresis, Stable Transfection, Expressing, Control

Journal: Orphanet Journal of Rare Diseases

Article Title: LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains

doi: 10.1186/1750-1172-9-68

Figure Lengend Snippet: Primary antibodies utilized for immunohistochemistry in the present

Article Snippet: HDAC6 , Santa Cruz Biotechnology , sc-11420 , rabbit , a peptide spanning amino acid residues 916-1215 of human HDAC6 , 0.8 μg/ml.

Techniques: Immunohistochemistry, Concentration Assay, Recombinant, Ubiquitin Proteomics, Isolation, Purification