Journal: Frontiers in Aging Neuroscience

Article Title: Class I HDAC Inhibitor Improves Synaptic Proteins and Repairs Cytoskeleton Through Regulating Synapse-Related Genes In vitro and In vivo

doi: 10.3389/fnagi.2020.619866

Figure Lengend Snippet: Effect of BG45 on the expression of HDAC1, HDAC2, synaptic-related proteins after the HF-488-Aβ 1–40 treatment. (A) Representative bands of the expression of HDAC1 and HDAC2 from control mice, HF-488-Aβ group and HF-488-Aβ + BG45 group. (B,C) Quantification of HDAC1 and HDAC2 is normalized to β-actin and is shown as a % of control. The expression of HDAC1, 2 was increased in the HF-488-Aβ group. Compared with the HF-488-Aβ group, the expression was significantly decreased in the HF-488-Aβ + BG45 group. (D) Representative bands of the expression of synapse-related protein from the control group, HF-488-Aβ group, and HF-488-Aβ + BG45 group. (E–G) Quantification of spinophilin, PSD-95, and synaptophysin (SYP) is normalized to β-actin and is shown as a % of control. The expression of spinophilin, PSD-95, and SYP was significantly decreased in the HF-488-Aβ group compared with the control group, while was improved in the HF-488-Aβ + BG45 group. n = 5. * p < 0.05, ** p < 0.01.

Article Snippet: Equivalent protein lysates (30 μg) were separated by 10% SDS–PAGE and blotted onto PVDF membranes (Millipore, USA); membranes were then blocked with 5% nonfat milk in Tris buffer at room temperature for 2 h. Western blotting was performed by incubation overnight at 4°C with the following antibodies: rabbit postsynaptic density protein 95 (PSD-95, 1:1,000, Abcam, ab18258), rabbit synaptophysin (SYP, 1:1,000, Abcam, ab32127), rabbit spinophilin (1:1,000, Cell Signaling Technology, 14136), rabbit tau (1:1,000, Cell Signaling Technology, 46687), rabbit p-tau (1:1,000, Cell Signaling Technology, 20194), rabbit HDAC1, rabbit HDAC2 (1:1,000, Cell Signaling Technology, 65816), mouse GAPDH (1:5,000, Proteintech, 60004-1) and rabbit β-actin (1:1,000, ABclone, AC026).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Histone Deacetylase Inhibitors Downregulate Calcium Pyrophosphate Crystal Formation in Human Articular Chondrocytes

doi: 10.3390/ijms23052604

Figure Lengend Snippet: Histone deacetylase inhibitors (HDACis) downregulated class II HDAC member expressions and upregulated the acetylation of histones 3 and 4 in transforming growth factor (TGF)-β1-treated chondrocytes. ( A , B ) Human primary chondrocytes (HC-a cells) were pretreated with different concentrations of trichostatin A (TSA) (100~800 nM) and vorinostat (SAHA) (100–1000 nM) for 30 min and then treated with 5 ng/mL TGF-β1 for 24 h. Total cellular proteins were collected, and protein expressions were detected by Western blotting. Ac, acetylated.

Article Snippet: Two kinds of class I and II HDACis, TSA and SAHA, were obtained from Union Biomed (Taipei, Taiwan); recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA); the anti-ANKH and anti-α-tubulin antibodies were obtained from Novus Biologicals (Littleton, CO, USA); the class I HDAC antibody sampler kit, class II HDAC antibody sampler kit, anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-histone H3, anti-acetyl-histone H3-Lys9, and anti-acetylated-lysine (Ac-K-103) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA); the anti-ENPP1, anti-TNAP, anti-acetyl-histone H4, anti-TGF-β receptor I (TGFBR1), and anti-TGF-β receptor II (TGFBR2) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP), and anti-rabbit IgG HRP antibodies were obtained from GeneTex International (Hsinchu City, Taiwan).

Techniques: Histone Deacetylase Assay, Western Blot

Journal:

Article Title: Modulation of NF-?B-dependent transcription and cell survival by the SIRT1 deacetylase

doi: 10.1038/sj.emboj.7600244

Figure Lengend Snippet: NAD-dependent HDACs regulate NF-κB transcription. (A) SIRT1 protein level in whole-cell extracts of NSCLC cell lines and normal lung epithelial cell lines (NL-20). (B) NSCLC cells were transiently transfected with the NF-κB-responsive reporter-3xκB luciferase. Cells were left untreated (No Add) or treated overnight with resveratrol (Res, 30 μM), nicotinamide (NAM, 500 μM), splitomicin (Split, 120 μM), or TSA (500 nM). TNFα (10 ng/ml) was added the next day for 8 h before harvesting the cells. All transfections were normalized with CMV-β-galactosidase activity. (C) Cell-based HDAC assays were performed by adding the Fluor de lys substrate alone (No Add) or in combination with resveratrol, nicotinamide, splitomicin, or TSA to the growth medium. Exogenous nicotinamide was added in vitro to untreated cell extracts (No Add+NAM) to ensure that the HDAC activity observed was NAD-dependent. Data represent a typical experiment performed in duplicate, with mean±SD (D) NSCLC cells were co-transfected with the 3xκB luciferase reporter, V5-tagged SIRT1, Myc-tagged HDAC3, or empty vector control (VC). Cells were treated with TNFα for 8 h before harvesting for luciferase activities. Western blot analysis confirmed the expression of V5-tagged SIRT1 and Myc-tagged HDAC3 proteins. (E) NSCLC cells were co-transfected with the 3xκB-luciferase reporter, or expression plasmids encoding SIRT1 protein or vector control. In addition, cells were transfected with p50, RelA/p65 expression vectors, or both.

Article Snippet: NSCLC cells were washed with PBS and incubated with phenol red-free complete media supplemented with 200 μM Fluor de Lys HDAC substrate (BIOMOL) only, or in the presence of resveratrol (30 μM), nicotinamide (500 μM), splitomicin (120 μM), or TSA (500 nM).

Techniques: Transfection, Luciferase, Activity Assay, In Vitro, Plasmid Preparation, Western Blot, Expressing

Journal:

Article Title: Modulation of NF-?B-dependent transcription and cell survival by the SIRT1 deacetylase

doi: 10.1038/sj.emboj.7600244

Figure Lengend Snippet: Resveratrol and SIRT1 inhibit the RelA/p65 transactivation domain by targeting lysine 310. (A) HEK 293T cells were co-transfected with plasmids encoding RelA/p65(1–313) and p300. Whole-cell extracts were immunoprecipitated and incubated with either recombinant SIRT1 or HDAC-defective mutant SIRT1(H363Y) protein in the presence or absence of NAD. (B) HEK 293T cells were co-transfected with expression plasmids as in (A). The transfected cells were left alone or treated with resveratrol (50 μM) or nicotinamide (500 μM) overnight. (C) H460 cells were co-transfected with the Gal4-luciferse reporter and Gal4-p65(286–551) or a mutant Gal4-p65(286–551, K310R). Additionally, cells were transfected with expression plasmids encoding SIRT1 or vector control. Fold repression was calculated where basal transcription values for either Gal4-p65+vector or Gal4-p65(K310R)+vector were normalized to 1.

Article Snippet: NSCLC cells were washed with PBS and incubated with phenol red-free complete media supplemented with 200 μM Fluor de Lys HDAC substrate (BIOMOL) only, or in the presence of resveratrol (30 μM), nicotinamide (500 μM), splitomicin (120 μM), or TSA (500 nM).

Techniques: Transfection, Immunoprecipitation, Incubation, Recombinant, Mutagenesis, Expressing, Plasmid Preparation

Journal: Molecules

Article Title: HDAC Inhibitory and Anti-Cancer Activities of Curcumin and Curcumin Derivative CU17 against Human Lung Cancer A549 Cells

doi: 10.3390/molecules27134014

Figure Lengend Snippet: HDAC inhibitory activity of CU17. ( a ) In vitro HDAC inhibitory activity of CU17 was analyzed by the Fluor-de-Lys HDAC Fluorometric Activity Assay Kit. Bar graphs were expressed as relative HDAC activity with respect to the control (DMSO). Trichostatin A (TSA) was used as a positive control. Each value represents the mean ± SD from three independent experiments. ( b ) Western blot analysis of hyperacetylation of histone H3 in A549 cells after exposure to varying concentrations of CU17 for 24 h. DMSO: ethanol (1:1, v / v ) and cisplatin (10 µM) were used as negative and positive controls, respectively. Total ERK1/2 was used as a loading control for Western blotting. ( c ) The relative fold of protein expression was calculated using the intensity of the protein band in comparison to a loading control and shown as a bar graph. Bar graph displayed the mean from three independent experiments. * p < 0.05 indicates a significant difference between the treatment and solvent control.

Article Snippet: Inhibition of HDAC activity in vitro was determined by using the Fluor-de-Lys HDAC Fluorometric Activity Assay Kit (Biomol, Enzo Life Sciences International, Inc., New York, NY, USA).

Techniques: Activity Assay, In Vitro, Control, Positive Control, Western Blot, Expressing, Comparison, Solvent

Journal: Scientific Reports

Article Title: New 5-Aryl-Substituted 2-Aminobenzamide-Type HDAC Inhibitors with a Diketopiperazine Group and Their Ameliorating Effects on Ischemia-Induced Neuronal Cell Death

doi: 10.1038/s41598-018-19664-9

Figure Lengend Snippet: Inhibitory activities of HDAC inhibitors against activation of HDAC1, 2, 3, 8 and 6.

Article Snippet: The inhibitory activities of compounds against HDAC1, 2, 3, 8 (Class I) and 6 (Class II) were measured utilizing the Fluorometric Drug Discovery Assay Kit BML-AK511 (HDAC1), BML-AK 512 (HDAC2), and BML-AK 531 (HDAC3), BML-SE145-0100 (HDAC8) and BML-SE508-0050 (HDAC6) (supplied from Enzo Life Sciences).

Techniques: Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Probing the interaction between the histone methyltransferase/deacetylase subunit RBBP4/7 and the transcription factor BCL11A in epigenetic complexes

doi: 10.1074/jbc.M117.811463

Figure Lengend Snippet: Pulldown assays show that BCL11A(2–16) peptide is sufficient to interact with PRC2, NuRD, and SIN3A epigenetic complexes in SUM149 cell lysate. A, amino acid sequences of the biotin-labeled BCL11A(2–16) WT and scramble (SCR) peptides used in our pulldown studies. B, Western blot analysis of RBBP4, RBBP7, HDAC1, HDAC2, and other components of PRC2 (EZH2 and SUZ12), NuRD (MTA1), SIN3A (SIN3A), and CoREST (CoREST) complexes following BCL11A(2–16) WT or scramble peptide pulldown. Ten micrograms of SUM149 whole-cell lysate (∼4.4% of the lysate used in the pulldown lanes) were loaded as a control for detection of the proteins of interest. C, HDAC activity assay following BCL11A(2–16) WT or scramble peptide pulldown was done using the HDAC fluorometric activity assay kit (Cayman Chemical). To determine the background fluorescence level, the pulldown samples were treated with 1 μm TSA, an HDAC inhibitor, before the assay. Results shown are mean ± S.D. (****, p < 0.0001, one-way analysis of variance). D, HDAC activity (nmol/min/ml) following BCL11A(2–16) WT or scramble peptide pulldown was calculated as described under “Materials and methods.” Results shown are mean ± S.D. (***, p < 0.001, Student's t test).

Article Snippet: C, HDAC activity assay following BCL11A(2–16) WT or scramble peptide pulldown was done using the HDAC fluorometric activity assay kit (Cayman Chemical).

Techniques: Labeling, Western Blot, Control, HDAC Activity Assay, Activity Assay, Fluorescence