Journal: Molecular & Cellular Proteomics : MCP

Article Title: Histone Deacetylase Inhibitors (HDACi) Cause the Selective Depletion of Bromodomain Containing Proteins (BCPs) *

doi: 10.1074/mcp.M114.042499

Figure Lengend Snippet: Determination of optimal drug treatment conditions ( A ) HeLa cells were cultivated for 24h and then treated with 1. 5 μ m TSA or 2 μ m Vorinostat for 48h. Control cells were treated with a respective amount of ethanol. Cells treated with HDACi show a typical morphology characterized by elongated protrusion. Scale bars are 100 μm. ( B ) MTT cell viability assay upon Vorinostat treatment. Vorinostat concentrations ranging from 0.2 μ m to 40 μ m were tested for three biological replicates. The data were expressed as percentage viability relative to untreated controls (set to 100%). ( C ) Western blotting of total cell lysate and nuclei with antibody against acetylated lysines (MW ∼18 kDa) shows an increase of acetylated histones following treatment with increasing TSA concentrations ranging between 0.5 μ m and 2 μ m . 50,000 cells or nuclei were loaded per lane, except the last two lanes of TSA treatment where only 28,000 cells were used. As a loading control an antibody against LaminB1/B2 (MW ∼66 kDa) was used. The red rectangle indicates a TSA concentration of 1.5 μ m that was used for all the further experiments. ( D ) Increase of acetylation on histone 3 (H3K18acK23ac) measured in nuclei, using shotgun proteomics, upon HDACi treatment. All shotgun experiments were carried out in triplicate. ( E ) Pie chart showing the proportion of significantly regulated proteins with a p value ≤ .01 and a log 2 fold change ≤ −1 or ≥ 1 in comparison to the total number of identified proteins upon TSA treatment. Major changes occur in the nuclear proteome while the majority of cytoplasmic proteins remain unaffected by the treatment. All shotgun experiments were carried out in triplicate. ( F ) Correlation of log 2 fold changes induced by Vorinostat and NaB on the nuclear proteome. The correlation was calculated using the Spearman's rank correlation coefficient. Purple dots indicate proteins that have a p value ≤ .01 in at least one of the two conditions and a log 2 fold change ≤ −1 or ≥ 1. The number of significant cases (“purple dots”) for each quadrant is indicated.

Article Snippet: Cells were allowed to attach to the culture dish for 24 h, then the drug, 2 μ m Vorinostat (LKT Laboratories, Inc., St. Paul, MN), 1.5 μ m TSA (Sigma-Aldrich), 300 m m sodium butyrate (Sigma-Aldrich) or 300 n m camptothecin (CPT) (Sigma-Aldrich), was added and cells were treated for 12 or 48 h. As a control, cells were treated with the same volume of ethanol or DMSO, in the case of CPT, for 12 or 48 h. The cells were washed twice with PBS for collection, while still attached to the culture dish, to exclude floating cells for all further experiments.

Techniques: Control, Viability Assay, Western Blot, Concentration Assay, Comparison

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Histone Deacetylase Inhibitors (HDACi) Cause the Selective Depletion of Bromodomain Containing Proteins (BCPs) *

doi: 10.1074/mcp.M114.042499

Figure Lengend Snippet: HDACi treatment causes a massive remodeling of bromodomain containing proteins ( A ) Gene ontology enrichment analysis of nuclear proteins affected by TSA treatment. Down-regulated proteins are significantly enriched in BCPs (fold enrichment ≥ 2, EASE Score (modified fisher exact p value) ≤ .1; only categories containing at least eight proteins are displayed). Enriched groups with a red star contain BCPs. ( B ) Heatmap showing the effect of HDACi and CPT treatment on BCPs. Three biological replicates per treatment were analyzed and displayed by their -log 10 p value and log 2 fold change. BCPs are grouped in functional groups. While the majority of the BCPs are significantly down-regulated, BCPs involved in protein degradation pathways are up-regulated by HDACi treatment. Orange squares in the p value heatmap indicate proteins that were identified exclusively and consistently in one sample group in all the three replicates but not in the other, and they were therefore considered as potentially regulated by the drug treatment. These cases were assigned an arbitrary log 2 fold change of +3 (for proteins identified only after drug treatment) or -3 (for proteins identified only in control samples). ( C ) Phylogenetic tree of BCPs based on bromodomain sequence similarity (adapted from ). While down-regulation is apparent in almost all classes of BCPs, the up-regulated BCPs group into two clusters formed by BRD2, BRD3, and BRD4, and by the three E3 ubiquitin ligase-containing BPCs, respectively. ( D ) Western blot of total cell lysate with antibody against p21 (MW ∼18 kDa) shows an increased abundance of p21 following treatment with TSA (1.5 μ m ), Vorinostat (2 μ m ) and NaB (300 m m ) already at 12 h (framed red). 50,000 cells were loaded per lane. As a loading control an antibody against glyceraldehyde-3-phosphate dehydrogenase (MW ∼36 kDa) was used. ( E ) Bar chart of the log 2 -fold changes of selected BCPs (CREBBP, BRD1, and PBRM1) as observed after HDACi and CPT treatment. BCPs depletion is induced by HDACi but not by CPT. All the reported values are averages of three biological replicates, and error bars indicate the standard error of the mean. Asterisks indicate significant cases. Cyan bars indicate proteins that were identified exclusively and consistently in control samples but were not detectable after drug treatment. These cases were considered as potentially regulated by HDACi, and they were assigned an arbitrary log 2 -fold change of −3.

Article Snippet: Cells were allowed to attach to the culture dish for 24 h, then the drug, 2 μ m Vorinostat (LKT Laboratories, Inc., St. Paul, MN), 1.5 μ m TSA (Sigma-Aldrich), 300 m m sodium butyrate (Sigma-Aldrich) or 300 n m camptothecin (CPT) (Sigma-Aldrich), was added and cells were treated for 12 or 48 h. As a control, cells were treated with the same volume of ethanol or DMSO, in the case of CPT, for 12 or 48 h. The cells were washed twice with PBS for collection, while still attached to the culture dish, to exclude floating cells for all further experiments.

Techniques: Modification, Functional Assay, Control, Sequencing, Ubiquitin Proteomics, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Histone Deacetylase Inhibitors (HDACi) Cause the Selective Depletion of Bromodomain Containing Proteins (BCPs) *

doi: 10.1074/mcp.M114.042499

Figure Lengend Snippet: The abundance of nuclear proteasome increases in response to both hydroxamate HDACis and the apoptosis-inducing drug CPT. The abundance of proteasome subunits increases dramatically in the nuclear fraction after treatment with TSA, Vorinostat, and CPT. Volcano plots of all proteins quantified in HeLa nuclei in comparison to their respective control for TSA ( A ), Vorinostat ( B ), and CPT (C) treatment. Purple dots indicate the identified subunits of the proteasome. ( D ) Comparison of the proteasomal distribution within subcellular fractionated cells upon HDACi treatment using shotgun mass spectrometry. The measured intensity of the proteasome subunits was normalized to the number of cells or nuclei analyzed. Dark gray bars indicate the cytosolic, light gray bars the nuclear fraction. The nuclear-localized proteasome increased up to 10-fold in abundance following HDACi treatment, while in the cytosol the abundance of the proteasome was reduced by 19 and 12% by TSA and Vorinostat, respectively. This indicates that the increase in nuclear-localized proteasome is mediated by relocalization of the proteasome from the cytosol to the nucleus following drug treatment.

Article Snippet: Cells were allowed to attach to the culture dish for 24 h, then the drug, 2 μ m Vorinostat (LKT Laboratories, Inc., St. Paul, MN), 1.5 μ m TSA (Sigma-Aldrich), 300 m m sodium butyrate (Sigma-Aldrich) or 300 n m camptothecin (CPT) (Sigma-Aldrich), was added and cells were treated for 12 or 48 h. As a control, cells were treated with the same volume of ethanol or DMSO, in the case of CPT, for 12 or 48 h. The cells were washed twice with PBS for collection, while still attached to the culture dish, to exclude floating cells for all further experiments.

Techniques: Comparison, Control, Mass Spectrometry

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: M344 HDAC selectivity profile

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques:

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Concentration response curves demonstrating selectivity profile of M344. Each compound is tested in duplicates in biochemical HDAC activity assays (BPS Biosciences). Each data point represents the mean percent activity ± SEM. M344 has submicromolar to low micromolar IC 50 values for HDACs 1, 2, 3, 6, 8, and 10.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Concentration Assay, Activity Assay

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Heat map summarizing up- and down-regulation of genes after M344 treatment of HEK/APP sw cells. Green indicates down-regulation of gene expression. Red indicates up-regulation. Changes are considered significant if FDR < 0.05, P < 0.05, and fold change > 1.2. n = 6. NanoString Data were analyzed using nSolver software 3.0.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Expressing, Software

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Summary of gene expression changes in HEK/APP sw cells after M344 treatment

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Expressing

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Effects of M344 on ADAM10, BACE1, and APP processing in HEK/APP sw cells. ( A ) RT-qPCR data showing significant increase of ADAM10 and ( B ) significant decrease of BACE1 after M344 treatment. ( C ) Representative Western blots of APP metabolites ADAM10 and BACE1 after M344 and garcinol treatments. Densitometry of bands from Western blots show significant increases in ( D ) sAPPα and ( E ) CTFα after M344 treatment. ( F ) There is a significant increase in the ADAM10 98kDa precursor compared with DMSO controls and ( G ) a significant decrease in BACE1 expression with M344. ( H ) M344 significantly increases immature APP and ( I ) decreases mature APP. All cells were treated with either 0.2% DMSO buffer or 10 μM of compounds in 0.2% DMSO. * P < 0.05, ** P < 0.01, **** P < 0.0001; n = 3; mean ± SEM.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Effects of different HDAC inhibitors on Aβ42/Aβ40 ratio and cell viability. ( A ) Several HDAC inhibitors significantly reduce Aβ42/Aβ40 ratio at 10 μM concentration. ( B ) M344 presents no effect on cell viability, whereas SAHA, oxamflatin, and trichostatin significantly reduce cell viability. All drugs were tested in duplicates. Mean ± SEM; *** P < 0.001, **** P < 0.0001.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Concentration Assay

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Representative Western blot of histone acetylation in HEK/APP sw cells. Purified histone extracts from cells treated with M344 for 48 h present significant increases of acetylation at ( A ) H3K27 and ( B ) H4K12 residues. n = 3; mean ± SEM; * P < 0.05, ** P < 0.01.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Western Blot, Purification

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Effects of M344 treatment of HEK/APP sw cells on histone acetylation over time. Purified histones from M344-treated HEK/APP sw cells show that M344 increases histone acetylation in a time-dependent manner as revealed by both increased H4K12 acetylation and increased pan-acetylation of lysine residues. n = 3; mean ± SEM; * P < 0.05, ** P < 0.01. ns, not significant.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Purification

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Representative Western blot of cell surface APP. The N termini of cell surface proteins were biotinylated on live HEK/APP sw cells with sulfosuccinimidyl-6-[biotin-amido]hexanoate (Sulfo-NHS-LC-Biotin; Thermo Fisher Scientific). Cell surface proteins were then captured with streptavidin beads, boiled in Laemmli buffer, separated by polyacrylamide gel electrophoresis, and transferred onto PVDF membranes. Hybridization of these membranes with anti-APP-CTF antibody (Calbiochem) shows that there is an accumulation of both mature and immature APP at the cell surface, further supporting an effect of M344 on APP trafficking. n = 4, mean ± SEM; ** P < 0.01.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Western Blot, Polyacrylamide Gel Electrophoresis, Hybridization

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Analysis of Aβ 1–42 , BACE1, ADAM10, and phospho-tau Ser 396 in the hippocampus of 3xTg AD mice. ( A ) M344 significantly reduces levels of Aβ 1–42 at 3 mg/kg, as determined by ELISA. ( B ) RT-qPCR results show that ADAM10 gene expression is significantly increased in the hippocampus of 3xTg mice treated with 10 mg/kg. ( C ) BACE1 mRNA level is significantly reduced in the hippocampus of mice treated at 3 mg/kg. ( D ) Both 3 mg/kg and 10 mg/kg of M344 significantly decrease tau phosphorylation at serine residue 396, as determined by ELISA. Mean ± SEM; * P < 0.05, ** P < 0.01. ns, not significant.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Representative Western blot of M344 treatment of CHO cells overexpressing wild-type APP. These data show that treatment of CHO/APP cells with M344 results in significant increase of sAPPα and CTFα, supporting that the effect of M344 is not cell-dependent, and that its effect on APP also occurs in the wild-type version of the protein. n = 3; mean ± SEM; * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Western Blot

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Gene expression levels of APP trafficking and neuroprotective genes after M344 treatment of HEK/APP sw cells. Treatment with 10 μM of M344 causes a significant increase in ( A ) FE65 and ( B ) MINT2. ( C ) RT-qPCR results show BDNF gene expression is significantly increased in the presence of 10 µM M344. ( D ) RT-qPCR data show that the overexpression of APP sw decreases REST levels below baseline, comparing HEK + DMSO versus HEK/APP sw + DMSO. Compound M344 significantly increases REST gene expression in HEK-293 cells and in HEK/APPsw cells. ( E ) Western blot showing that M344 significantly increases BDNF protein level. n = 3–6; mean ± SEM; * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Western Blot

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Gene expression of BDNF gene expression in different cells. Treatment of HEK-293, SH-SY5Y, and N2A cells with 10 μM of M344 significantly increases BDNF gene expression, demonstrating that effects are not cell-type-specific. n = 6; mean ± SEM; ** P < 0.01, **** P < 0.0001.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Expressing

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Pharmacokinetics and histone acetylation after M344 treatment of wild-type mice. ( A ) I.p. injection of 10 mg/kg of M344 results in significant increase of M344 brain concentration after 15 min of treatment. ( B ) The same treatment also causes significant increase of M344 plasma concentrations after 15 min. ( C ) Representative Western blots from purified histone extracts show increased acetylation of H4K12 in the frontal cortex but not in the cerebellum after i.p. injection with 10 mg/kg of M344 for 30 min. ( D ) Quantification of frontal cortex Western blots from purified histone extracts shows significant increase in H4K12 acetylation after M344 treatment. ( E ) Quantification of cerebellum Western blots shows there is no significant difference in acetylation at the H4K12 residue in purified histone extracts from the cerebellum after M344 treatment. ( F ) Summary of M344 free fraction, free concentration, and brain/plasma ratio levels after 10 mg/kg i.p. treatment. n = 3; mean ± SEM; * P < 0.05, **** P < 0.0001. ns, not significant.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Injection, Concentration Assay, Western Blot, Purification

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Effects of M344 treatment on behavior of the 3xTg AD mice. ( A ) I.p. injection of M344 increases Y-maze spontaneous alternation in mice at both 3 mg/kg and 10 mg/kg, with ( B ) showing no significant differences in total arm entries. ( C ) Open field test shows that animals treated with M344 have no locomotion deficits and ( D ) travel at similar velocity compared with controls. ( E ) Injection of 3 mg/kg and 10 mg/kg of M344 increases novel object recognition performance of mice as determined by duration of exploration and ( F ) frequency of novel object exploration. ( G ) Barnes maze acquisition trials for these mice show significantly fewer errors in trial 3 for mice treated with 10 mg/kg of M344 and in trial 5 for mice treated with 3 mg/kg or 10 mg/kg. ( H ) Barnes maze probe trial shows that M344 significantly increases spatial memory as determined by decreased errors in treated mice. Vehicle: n = 10; 3 mg/kg: n = 9; 10 mg/kg: n = 8; mean ± SEM; * P < 0.05, ** P < 0.01. ns, not significant.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques: Injection

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

Article Title: M344 promotes nonamyloidogenic amyloid precursor protein processing while normalizing Alzheimer’s disease genes and improving memory

doi: 10.1073/pnas.1707544114

Figure Lengend Snippet: Effects of M344 on α-tubulin acetylation in HEK/APP sw cells. Treatment of HEK/APP sw cells with 10 μM of M344 results in a significant increase of the α-tubulin acetylation, an HDAC6 target. n = 3; mean ± SEM; * P < 0.05.

Article Snippet: The selectivity profile of M344 was determined biochemically by performing activity assays in duplicate with each of the 11 zinc-dependent HDACs at 10-point 1:3 dilutions, starting at 100 μM (BPS Biosciences).

Techniques:

Journal: Frontiers in Oncology

Article Title: Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant

doi: 10.3389/fonc.2022.959133

Figure Lengend Snippet: Combined treatment of vorinostat and PARPis enhanced the decrease in PED-GBM survival: SU-DIPG-VI cells (A, B) and SU-DIPG-IV cells (C, D) were plated in triplicates at a density of 11,550 or 3,300 cells/cm 2 , respectively. Drugs were added 24 h post-plating. KNS-42 were plated either for clonogenic assay (E) or for survival assay (F) at a density of 55 or 6,600 cells/cm 2 , respectively. Values are mean survival (%) ± S.D relative to control. Differences among all experimental groups, as well as between each experimental group and control, were significant - *p< 0.5, **p< 0.005. (G–I) . Cells were processed for Western blot analysis of treatment-induced changes in the ratio of peIF2α/eIF2α. Numbers at the bottom of the autoradiograms indicate changes of peIF2α/eIF2α and differences in loaded proteins (Ponceau) relative to control. (J, K) . SU-DIPG VI and HAs cells were plated in triplicates at the same density (3,300 cells/cm 2 ) and treated with drugs 24 h post-plating. Values are the mean number of cell division or cell death (%) ± S.D relative to control. Differences between treated and untreated experimental groups, as well as between SU-DIPG-VI and HAs, were significant *p< 0.05, **p< 0.005. V—vorinostat, Ola—Olaparib, Vel—veliparib, Nir—niraparib. The experiments were reproduced at least twice with similar results.

Article Snippet: Vorinostat was obtained from LC Laboratories (Boston, MA), olaparib and niraparib were obtained from MedChemExpress (Monmouth Junction, NJ), salubrinal was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), raphin-1 was obtained from Tocris Bioscience (Bristol, UK), and veliparib was obtained from APExBIO (Houston, TX).

Techniques: Clonogenic Assay, Clonogenic Cell Survival Assay, Control, Western Blot

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Inhibition of Histone Deacetylases

doi: 10.1385/1-59259-828-5:087

Figure Lengend Snippet: Human histone deacetylase subunits

Article Snippet: HDAC inhibitors: SAHA (suberoylanilide hydroxamic acid, vorinostat, Cayman); TSA (7-[4-(dimethylamino) phenyl]- N -hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; Sigma); MS275 (Selleck Chemicals); belinostat (PXD101, Selleck Chemicals); and panobinostat (LBH589, Selleck Chemicals).

Techniques: Histone Deacetylase Assay

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Inhibition of Histone Deacetylases

doi: 10.1385/1-59259-828-5:087

Figure Lengend Snippet: Characteristics of some HDAC inhibitors in clinical trials

Article Snippet: HDAC inhibitors: SAHA (suberoylanilide hydroxamic acid, vorinostat, Cayman); TSA (7-[4-(dimethylamino) phenyl]- N -hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; Sigma); MS275 (Selleck Chemicals); belinostat (PXD101, Selleck Chemicals); and panobinostat (LBH589, Selleck Chemicals).

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Inhibition of Histone Deacetylases

doi: 10.1385/1-59259-828-5:087

Figure Lengend Snippet: Techniques for studying HDAC inhibition in human breast cancer. After human breast cancer cells are treated with HDAC inhibitors, immunological detection methods such as Western blot or immunochemistry can be used to determine the level of histone acetylation using specific antibodies against histone H3 or H4 or specific histone lysine residues such as AcH3K9, AcH3K27, and AcH4K20. Chromatin immunoprecipitation (ChIP) is used to determine the interaction of site-specific acetylated histones with promoters of genes of interest in breast cancer cells after HDAC inhibitor treatment. DNA sequences bound to a particular acetylated histone or nonhistone protein can be isolated by ChIP and these fragments can subsequently be hybridized to a DNA microarray (such as a tiling array). This so-called ChIP-on-chip technology allows the determination of acetylated histone binding occupancy throughout the genome of the cancer cell. Quantitative-PCR is able to precisely measure the specific gene expression changes in the presence of HDAC inhibitor treatment. Microarray-based gene expression profiling can be used to identify genes whose expression is altered by HDAC inhibitor treatment. Several mass spectrometry (MS)-based proteomic methods exist to quantitatively analyze proteins that are hyperacetylated after treatment with HDAC inhibitors as well as determine isoform specific occupancy of histone modifications.

Article Snippet: HDAC inhibitors: SAHA (suberoylanilide hydroxamic acid, vorinostat, Cayman); TSA (7-[4-(dimethylamino) phenyl]- N -hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; Sigma); MS275 (Selleck Chemicals); belinostat (PXD101, Selleck Chemicals); and panobinostat (LBH589, Selleck Chemicals).

Techniques: Inhibition, Western Blot, Chromatin Immunoprecipitation, Isolation, Microarray, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Mass Spectrometry

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Inhibition of Histone Deacetylases

doi: 10.1385/1-59259-828-5:087

Figure Lengend Snippet: RT-PCR primers and conditions for genes reactivated by HDAC inhibitors in human breast cancer cells

Article Snippet: HDAC inhibitors: SAHA (suberoylanilide hydroxamic acid, vorinostat, Cayman); TSA (7-[4-(dimethylamino) phenyl]- N -hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; Sigma); MS275 (Selleck Chemicals); belinostat (PXD101, Selleck Chemicals); and panobinostat (LBH589, Selleck Chemicals).

Techniques:

Journal: Scientific Reports

Article Title: Histone deacetylase inhibitors suppress ACE2 and ABO simultaneously, suggesting a preventive potential against COVID-19

doi: 10.1038/s41598-021-82970-2

Figure Lengend Snippet: Alterations in the expression of ABO , ACE2 and TMPRSS2 , cell proliferation, and viability by treatment of KATOIII cells with HDACIs. ( A – O ) Each panel represents the relative amount of ABO ( A , D , G , J and M ), ACE2 ( B , E , H , K and N ) or TMPRSS2 ( C , F , I , L and O ) transcripts in KATOIII cells incubated with or without various concentrations of sodium butyrate ( A – C ), panobinostat ( D – F ), sodium valproate ( G – I ), vorinostat ( J – L ) or trichostatin A ( M – O ) for 24 h. The amounts are expressed as mean fold values relative to those without HDACI. The asterisks represent a significant reduction compared to the values without HDACI ( p < 0.05). ( P – T ) Each panel represents both the proliferation and viability of KATOIII cells incubated with or without various concentrations of HDACIs for 24 h. The bars indicate cell proliferation as the number of live cells relative to that without HDACI treatment on the left y-axes, while the dots and lines denote the absolute cell viability on the right y-axes.

Article Snippet: The HDMCIs we used included sodium butyrate (#303410; Sigma-Akdrich), panobinostat (#13280; Cayman Chemical Company), sodium valproate (#13033; Cayman Chemical Company), vorinostat (#10009929; Cayman Chemical Company) and trichostatin A (#89730; Cayman Chemical Company).

Techniques: Expressing, Incubation