Journal: bioRxiv

Article Title: Overcome Prostate Cancer Resistance to Immune Checkpoint Therapy with Ketogenic Diet-Induced Epigenetic Reprogramming

doi: 10.1101/2023.08.07.552383

Figure Lengend Snippet: (A) Flow cytometry histograms of H2-K b and H2-D b for the four cell lines. (B) Volcano plot for differentially expressed genes in PPS-ICBR cells treated with 5 µM vorinostat or DMSO for 12h. Cutoff thresholds and two MHC-I genes were indicated. (C) Flow cytometry histograms of H2-K b for PPS-ICBR cells treated with 5 µM vorinostat or DMSO for 12h. (D) H3K9Ac ChIP-qPCR of the H2-K1 locus in PPS-ICBR cells treated with 5µM vorinostat or DMSO. N=3/condition. (E) Top enriched KEGG pathways for genes upregulated or downregulated by vorinostat in PPS-ICBR cells. (F) Flow cytometry plot of H2-K b and H2-D b for PPS-ICBR cells transduced with control shRNA (scScr) or Hdac1 shRNA (shHdac1). (G) Individual growth curves of PPS-ICBR tumors in mice treated with vehicle (n=20), vorinostat (n=10), ICB (anti-PD1+anti-CTLA4, n=10), or ICB+vorinostat (n=24). Complete response ratios were labeled. (H) Western blot of α-tub-K40Ac and β-actin of PPS-ICBR cells or tumors treated with vehicle or vorinostat. (I) Western blot of H3K9Ac, total H3 and β-actin of PPS-ICBR tumors treated with vehicle, vorinostat, ICB, or ICB+vorinostat. (J) Flow cytometry histograms of H2-K b for PCa cells in the dissociated PPS-ICBR tumors treated with vehicle, vorinostat, ICB, or ICB+vorinostat. (K) Quantification of CD8α IHC signals for PPS-ICBR tumors in mice treated with vehicle, vorinostat, ICB, ICB+vorinostat, or ICB+vorinostat+anti-CD8. N=7/group. (L) Growth curves of PPS-ICBR tumors in mice treated with vehicle (n=8), ICB+vorinostat+anti-CD8 (n=8), or ICB+vorinostat+isotype (n=5). Arrows indicate the dosing of anti-CD8 or isotype IgG. (M) Individual growth curves of PPS-ICBR tumors (n=6, no growth) or MC38 tumors (n=2, both grew) in C57BL/6 hosts previously cured of PPS-ICBR tumors with ICB+vorinostat treatment. In (D), (K) and (L), data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant; unpaired t-test for (D), Mann-Whitney test for (K), two-way ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparison test for (L).

Article Snippet: When tumors reached 50-100mm , mice were randomized to receive therapeutics, including anti-PD1 (BioLegend, 114116) and anti-CTLA4 (BioLegend, 106207) at 10mg/kg each i.p. twice a week, or vorinostat (MedChemExpress, HY-10221) at 25 mg/kg i.p. daily.

Techniques: Flow Cytometry, ChIP-qPCR, Transduction, Control, shRNA, Labeling, Western Blot, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Overcome Prostate Cancer Resistance to Immune Checkpoint Therapy with Ketogenic Diet-Induced Epigenetic Reprogramming

doi: 10.1101/2023.08.07.552383

Figure Lengend Snippet: (A) Schematic of the interim cohorts of vorinostat and/or ICB treatments for CyTOF. (B) Growth curves of PPS-ICBR tumors in mice treated with vehicle (n=4), vorinostat (n=4), ICB (n=6), and ICB+vorinostat (n=8). (C) CyTOF tSNE plots showing the 14 cell clusters annotated for the 4 conditions. (D) Cell cluster percentages plotted for the 4 conditions. (E) Cell cluster densities projected on the tSNE maps for the 4 conditions. (F) Percentages of tumor-infiltrating T cell subsets in the 4 conditions. (G) Percentages of tumor-infiltrating myeloid subsets in the 4 conditions. In (B), (F) and (G), data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ns, not significant; two-way ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparison test for (B), one-way ANOVA with Tukey’s multiple comparison test for (F) and (G).

Article Snippet: When tumors reached 50-100mm , mice were randomized to receive therapeutics, including anti-PD1 (BioLegend, 114116) and anti-CTLA4 (BioLegend, 106207) at 10mg/kg each i.p. twice a week, or vorinostat (MedChemExpress, HY-10221) at 25 mg/kg i.p. daily.

Techniques: Comparison

Journal: bioRxiv

Article Title: Overcome Prostate Cancer Resistance to Immune Checkpoint Therapy with Ketogenic Diet-Induced Epigenetic Reprogramming

doi: 10.1101/2023.08.07.552383

Figure Lengend Snippet: (A) Western blot of H3K9Ac and β-actin of PPS-ICBR cells treated with 5µM vorinostat, 3mM BHB and DMSO control for 12h. (B) Flow Cytometry of histograms of H2-K b for PPS-ICBR cells treated as indicated for 12h. (C) Caloric composition of SD and KD used in the study. (D) Schedule of CKD feeding. (E) Serum BHB level for C57BL/6 mice fed with SD (n=3) or CKD (n=3) for 3 cycles with BHB measured at cycle 3 day 5 and day 7. (F) Growth curves of PPS-ICBR tumors in C57BL/6 mice fed with SD (n=14) or CKD (n=10). (G) Western blot of H3K9Ac and α-tubulin for PPS-ICBR tumors from mice fed with SD or CKD. (H) Flow cytometry of H2-K b and H2-D b for PCa cells in the dissociated PPS-ICBR tumors from mice fed with SD or CKD. (I) Tumor growth curves of PPS-ICBR subcutaneous tumors in immunocompromised Batf3 -/- or Rag1 -/- C57BL/6 mice fed either a SD or CKD. ( Batf3 -/- SD N=8 CKD N=8. Rag1 -/- SD N=6 CKD N=10) (J) The rationale for Bdh1 knockout to abolish BHB from hepatic ketogenesis. (K) Western blot of Bdh1 and α-tubulin for liver tissue from wild type (WT) or Bdh1 -/- mice fed with SD, CKD, or BD. (L) Serum BHB levels from WT and Bdh1 -/- mice fed with SD or CKD (n=3∼4). (M) Volumes of PPS-ICBR tumors grown in WT mice fed with SD (n=5) or CKD (n=8) or Bdh1 -/- fed with CKD (n=6). (N) Survival curves of PPS-ICBR-bear mice treated as indicated (n=8/group). Tumor volumes at 1000mm were recorded as the endpoint. (O) Survival curves of PPS-ICBR-bear mice treated as indicated, SD (n=8), BD (n=10), SD+ICB (n=8), or BD+ICB (n=7). Tumor volumes at 1000mm were recorded as the endpoint. (P) Serum BHB levels in PPS-ICBR-bearing mice fed with SD (n=3) or BD (n=3). (Q) Flow cytometry median fluorescence intensity (MFI) of H2-K b for PCa cells in the dissociated PPS-ICBR tumors from mice treated with SD, BD, SD+ICB or BD+ICB (n=3/group). (R) Western blot of H3K9Ac, total H3 and α-tubulin for PPS-ICBR tumors from mice treated as indicated, with PPS-ICBR cells treated with 5uM vorinostat used as the control lane. In (E) (F) (I) (L) (M) (P) (Q), data represent mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, not significant; two-way ANOVA with Tukey’s multiple comparison test for (E), Mann-Whitney test for (F) (I), one-way ANOVA with Tukey’s multiple comparison test for (L) (M) (Q), log-rank test for (N) (O), unpaired t-test for (P).

Article Snippet: When tumors reached 50-100mm , mice were randomized to receive therapeutics, including anti-PD1 (BioLegend, 114116) and anti-CTLA4 (BioLegend, 106207) at 10mg/kg each i.p. twice a week, or vorinostat (MedChemExpress, HY-10221) at 25 mg/kg i.p. daily.

Techniques: Western Blot, Control, Flow Cytometry, Knock-Out, Fluorescence, Comparison, MANN-WHITNEY

Journal: Oncology reports

Article Title: Vorinostat enhances protein stability of p27 and p21 through negative regulation of Skp2 and Cks1 in human breast cancer cells.

doi: 10.3892/or.2012.1758

Figure Lengend Snippet: Figure 1. Effect of vorinostat on BrdU incorporation in MDA-MB-231 and MCF-7 human breast cancer cells. MDA-MB-231 (A) and MCF-7 (B) cells were treated with the IC50 of vorinostat (5.0 and 2.5 µM, respectively) for 72 h. Representative micrographs for BrdU staining at 24 h after treatment with vorinostat are shown. BrdU-labeling indices were determined at the indicated time points as described in Materials and methods. Columns indi cate the percent of BrdU-positive cells in five fields of 1,000 cells total from each separate experiment. *P<0.05 and **P<0.01 as compared with untreated control cells at each time point.

Article Snippet: Vorinostat (suberoylanilide-hydroxamic acid) was obtained from Selleck (Houston, TX).

Techniques: BrdU Incorporation Assay, BrdU Staining, Labeling, Control

Journal: Oncology reports

Article Title: Vorinostat enhances protein stability of p27 and p21 through negative regulation of Skp2 and Cks1 in human breast cancer cells.

doi: 10.3892/or.2012.1758

Figure Lengend Snippet: Figure 2. Effect of vorinostat on p27 and p21 expression in human breast cancer cells. (A and B) mRNA expression of p27 and p21 was analyzed by real-time PCR and was normalized to the level of β-actin, and the expres sion level is shown relative to the control as 1. Data represent the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 as compared with untreated control cells. (C) Lysates were prepared from MDA-MB-231 and MCF-7 cells at the indicated time points after vorinostat treatment. Western blot analysis was performed by using antibodies specific to the indicated proteins.

Article Snippet: Vorinostat (suberoylanilide-hydroxamic acid) was obtained from Selleck (Houston, TX).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Oncology reports

Article Title: Vorinostat enhances protein stability of p27 and p21 through negative regulation of Skp2 and Cks1 in human breast cancer cells.

doi: 10.3892/or.2012.1758

Figure Lengend Snippet: Figure 3. Effect of vorinostat on Skp2 and Cks1 expression in human breast cancer cells. (A and B) mRNA expression of Skp2 and Cks1 was analyzed by real-time PCR analysis and normalized to the level of β-actin. The expres sion level is shown relative to the control as 1 (A and B). Data represent the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 as compared with untreated control cells. (C) Lysates were prepared from MDA-MB-231 and MCF-7 cells at the indicated time points after vorinostat treatment. Western blot analysis was performed with antibodies specific to the indicated proteins.

Article Snippet: Vorinostat (suberoylanilide-hydroxamic acid) was obtained from Selleck (Houston, TX).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Oncology reports

Article Title: Vorinostat enhances protein stability of p27 and p21 through negative regulation of Skp2 and Cks1 in human breast cancer cells.

doi: 10.3892/or.2012.1758

Figure Lengend Snippet: Figure 4. Vorinostat enhances p27 and p21 protein stability in breast cancer cells. (A and B) MDA-MB-231 and (C and D) MCF-7 cells were treated with the IC50 of vorinostat (5.0 and 2.5 µM, respectively) for 24 h, and then 10 µg/ml CHX was added. (A and C) At the indicated time points after CHX treatment, cell lysates were prepared and western blot analysis was performed with antibodies specific to the indicated proteins. (B and D) Band intensity was normalized to actin, and the protein level is shown relative to the t=0 controls. Data represent the mean ± SD of three independent experiments. *P<0.05 and **P<0.01 as compared with control cells at each time point.

Article Snippet: Vorinostat (suberoylanilide-hydroxamic acid) was obtained from Selleck (Houston, TX).

Techniques: Western Blot, Control

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: Technology in cancer research & treatment

Article Title: Role of lncRNA AGAP2-AS1 in Breast Cancer Cell Resistance to Apoptosis by the Regulation of MTA1 Promoter Activity.

doi: 10.1177/15330338221085361

Figure Lengend Snippet: Figure 5. H3K27 upregulation enhances MTA1 expression pattern to promote BC cell resistance to apoptosis after AGAP2-AS1 silencing. MCF-7 cells in the si-AGAP2-AS1#2 were treated with M344, with DMSO treatment as the control. A, H3K27ac recruitment in MTA1 promoter of MCF-7 cells was detected by ChIP. B and C (the samples derived from the same experiment, and gels and blots were cropped and processed in parallel), MTA1 expression pattern in MCF-7 cells was determined by RT-qPCR (B) and western blot analysis (C). D, cell proliferation was evaluated via CCK-8 method. E and F, MCF-7 cell apoptosis was measured by flow cytometry (Annexin V-FITC and PI staining) (E) and TUNEL staining (200×) (F). Independent experiments were conducted 3 times independently. The results were presented as mean ± standard deviation. One-way ANOVA was used to analyze the data in panels A, B, C, E and F, two-way ANOVA was used to analyze the data in panel D. Tukey’s multiple comparisons test was applied for the post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: The MCF-7 cells were supplemented with 0.1 dimethyl sulphoxide (HY-Y0320, MedChemExpress Co., Ltd, Monmouth Junction, NJ, USA) or 10 μM M344 (a histone deacetylase inhibitor) (HY-13506, MedChemExpress Co., Ltd) for 48 h for subsequent experimentation.

Techniques: Expressing, Control, Derivative Assay, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Cytometry, Staining, TUNEL Assay, Standard Deviation

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: