Structured Review

Millipore hdac inhibitors
Histone deacetylase <t>(HDAC)</t> inhibitors reduce the expression of G6P and PCK1 in HepG2 cells. Cells were treated with <t>VPA</t> (( A , F ), 0.1, 1.0, and 10 mmol/L); SAHA (( B , G ), 0.1, 1.0, and 10 µmol/L); TSA ( C , H , 0.1, 0.3, and 1.0 µmol/L); MS275 (( D , I ), 1.0, 10, and 100 µmol/L); and MC1568 (( E , J ), 0.1, 1.0, and 10 µmol/L) for 48 h under hyperglycemic conditions. Expression of G6P or PCK1 was quantified by RT-qPCR. Cultivation under hyperglycemic conditions increased the expression of G6P or PCK1 , an effect that was attenuated by treatment with the pan-HDAC inhibitors VPA, SAHA, and TSA, as well as the HDAC class I-specific inhibitor, MS275, and the HDAC class IIa-specific inhibitor, MC1568. The graphs show the mean ± SEM of three independent experiments (* p
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Images

1) Product Images from "Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats"

Article Title: Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113539

Histone deacetylase (HDAC) inhibitors reduce the expression of G6P and PCK1 in HepG2 cells. Cells were treated with VPA (( A , F ), 0.1, 1.0, and 10 mmol/L); SAHA (( B , G ), 0.1, 1.0, and 10 µmol/L); TSA ( C , H , 0.1, 0.3, and 1.0 µmol/L); MS275 (( D , I ), 1.0, 10, and 100 µmol/L); and MC1568 (( E , J ), 0.1, 1.0, and 10 µmol/L) for 48 h under hyperglycemic conditions. Expression of G6P or PCK1 was quantified by RT-qPCR. Cultivation under hyperglycemic conditions increased the expression of G6P or PCK1 , an effect that was attenuated by treatment with the pan-HDAC inhibitors VPA, SAHA, and TSA, as well as the HDAC class I-specific inhibitor, MS275, and the HDAC class IIa-specific inhibitor, MC1568. The graphs show the mean ± SEM of three independent experiments (* p
Figure Legend Snippet: Histone deacetylase (HDAC) inhibitors reduce the expression of G6P and PCK1 in HepG2 cells. Cells were treated with VPA (( A , F ), 0.1, 1.0, and 10 mmol/L); SAHA (( B , G ), 0.1, 1.0, and 10 µmol/L); TSA ( C , H , 0.1, 0.3, and 1.0 µmol/L); MS275 (( D , I ), 1.0, 10, and 100 µmol/L); and MC1568 (( E , J ), 0.1, 1.0, and 10 µmol/L) for 48 h under hyperglycemic conditions. Expression of G6P or PCK1 was quantified by RT-qPCR. Cultivation under hyperglycemic conditions increased the expression of G6P or PCK1 , an effect that was attenuated by treatment with the pan-HDAC inhibitors VPA, SAHA, and TSA, as well as the HDAC class I-specific inhibitor, MS275, and the HDAC class IIa-specific inhibitor, MC1568. The graphs show the mean ± SEM of three independent experiments (* p

Techniques Used: Histone Deacetylase Assay, Expressing, Quantitative RT-PCR

2) Product Images from "Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes"

Article Title: Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes

Journal: Haematologica

doi: 10.3324/haematol.2008.001933

The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top
Figure Legend Snippet: The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top

Techniques Used: Immunostaining, Staining

3) Product Images from "Class I HDAC Inhibition Stimulates Cardiac Protein SUMOylation Through a Post-Translational Mechanism"

Article Title: Class I HDAC Inhibition Stimulates Cardiac Protein SUMOylation Through a Post-Translational Mechanism

Journal: Cellular signalling

doi: 10.1016/j.cellsig.2014.09.005

HDAC inhibition stimulates SUMOylation in cardiac cells. (A) Neonatal rat ventricular myocytes (NRVMs) were treated with the pan-HDAC inhibitor trichostatin A (TSA) for the indicated amounts of time. SUMO-1 conjugates were examined by immunoblotting.
Figure Legend Snippet: HDAC inhibition stimulates SUMOylation in cardiac cells. (A) Neonatal rat ventricular myocytes (NRVMs) were treated with the pan-HDAC inhibitor trichostatin A (TSA) for the indicated amounts of time. SUMO-1 conjugates were examined by immunoblotting.

Techniques Used: Inhibition

4) Product Images from "H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion by activating TRNP1 expression"

Article Title: H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion by activating TRNP1 expression

Journal: bioRxiv

doi: 10.1101/2021.03.06.434209

HDAC inhibition increase the genesis of BPs in dose-dependent fashion. (A) Immunofluorescence micrographs showing the quantity and distribution of PAX6 and TBR2 at E18.5 in control and BAF155cKO cortices, with or without two regimens of HDACi treatment. (B) Statistical analysis for dorsal cortical area revealed that more PAX6+ BPs and TBR2+ BPs were found in E18.5 cortex treated with TSA for E13.5-E17.5 than those in those injected with TSA for E13.5-E15.5. Notably, the TSA treatment also led to increased number of PAX6+vRGs at E18.5. (C) Proportions of cortical progenitors expressing PAX6 and/or TBR2 in VZ, SVZ, and IZ from dorsal area of WT+ Veh, WT+ TSA and BAF155cKO+ TSA cortex at E16.5. Values are presented as mean ± SEM (*P
Figure Legend Snippet: HDAC inhibition increase the genesis of BPs in dose-dependent fashion. (A) Immunofluorescence micrographs showing the quantity and distribution of PAX6 and TBR2 at E18.5 in control and BAF155cKO cortices, with or without two regimens of HDACi treatment. (B) Statistical analysis for dorsal cortical area revealed that more PAX6+ BPs and TBR2+ BPs were found in E18.5 cortex treated with TSA for E13.5-E17.5 than those in those injected with TSA for E13.5-E15.5. Notably, the TSA treatment also led to increased number of PAX6+vRGs at E18.5. (C) Proportions of cortical progenitors expressing PAX6 and/or TBR2 in VZ, SVZ, and IZ from dorsal area of WT+ Veh, WT+ TSA and BAF155cKO+ TSA cortex at E16.5. Values are presented as mean ± SEM (*P

Techniques Used: Inhibition, Immunofluorescence, Injection, Expressing

HDAC inhibition causes H3K9ac-linked upregulation of gene expression in BAF155cKO developing cortex, TBR2+ BPs and TBR2-cells. (A, B) H3K9ac is increased at loci of upregulated genes (A), but not at those of downregulated genes (B) in TSA treated BAF155cKO cortex. (C, D) Graphs of qPCR to examine gene expression (C) and ChIP-qPCR to quantify H3K9ac level (D). TSA treatment leads to increase in H3K9ac levels at promoters of neurogenesis-related genes (D) and their expression (C) both in WT and in BAF155cKO. ChIP-qPCR and qPCR results for a selection of genes are shown (WT_Veh: n = 3, WT_TSA: n = 4, BAF155 cKO_Veh: n = 4, BAF155 cKO_TSA: n = 4; general TSA effect: mixed linear model p -value
Figure Legend Snippet: HDAC inhibition causes H3K9ac-linked upregulation of gene expression in BAF155cKO developing cortex, TBR2+ BPs and TBR2-cells. (A, B) H3K9ac is increased at loci of upregulated genes (A), but not at those of downregulated genes (B) in TSA treated BAF155cKO cortex. (C, D) Graphs of qPCR to examine gene expression (C) and ChIP-qPCR to quantify H3K9ac level (D). TSA treatment leads to increase in H3K9ac levels at promoters of neurogenesis-related genes (D) and their expression (C) both in WT and in BAF155cKO. ChIP-qPCR and qPCR results for a selection of genes are shown (WT_Veh: n = 3, WT_TSA: n = 4, BAF155 cKO_Veh: n = 4, BAF155 cKO_TSA: n = 4; general TSA effect: mixed linear model p -value

Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Selection

Enhanced acetylation of H3 increases the number of basal progenitors (A) Experimental paradigm in which BAF155cKO and control embryos were treated with HDAC inhibitors (HDACi: TSA, SAHA, VPA) at the indicated developmental stages. (B) IHC images showing TBR2 (BPs), PAX6 (APs, BPs), and AP2 γ (APs, BPs) in sections from control and BAF155cKO embryos with or without TSA treatment. (C) Statistical comparisons indicate that increased H3 acetylation enhanced the number of BPs (TBR2+, PAX6+, AP2 γ + cells of the IZ) in TSA-treated WT and BAF155cKO embryos compared to vehicle (Veh)-treated controls. (D‒F) Confirmation of BPs/bRGs (arrows) based on their morphology. BPs/bRGs exhibit long basal processes but no apical processes in TSA-treated cortex, as revealed by double labeling of Pax6 with either DiI (D), or retroviral GFP (E), or pVIM (F). Notably, fanned fibers of PAX6+ BPs/bRGs were observed when DiI labeling was applied at the pia (D). (F) High-magnification image of a PAX6+/pVIM+ BPs/bRG, indicated by a red arrow. (G) Statistical analysis comparing number of PAX6+/pVIM+ BPs/bRGs with basal processes in the SVZ/IZ of the indicated embryos. (H, I) IHC analysis shows the expression of the human-enriched bRG markers, PTPRZ1 (H), and TNC (I), in PAX6+/TBR2- cells (arrows) of TSA-treated BAF155cKO cortex at E16.5 (see also fig. S8 for E18.5 cortex). Values are presented as means ± SEMs (* p
Figure Legend Snippet: Enhanced acetylation of H3 increases the number of basal progenitors (A) Experimental paradigm in which BAF155cKO and control embryos were treated with HDAC inhibitors (HDACi: TSA, SAHA, VPA) at the indicated developmental stages. (B) IHC images showing TBR2 (BPs), PAX6 (APs, BPs), and AP2 γ (APs, BPs) in sections from control and BAF155cKO embryos with or without TSA treatment. (C) Statistical comparisons indicate that increased H3 acetylation enhanced the number of BPs (TBR2+, PAX6+, AP2 γ + cells of the IZ) in TSA-treated WT and BAF155cKO embryos compared to vehicle (Veh)-treated controls. (D‒F) Confirmation of BPs/bRGs (arrows) based on their morphology. BPs/bRGs exhibit long basal processes but no apical processes in TSA-treated cortex, as revealed by double labeling of Pax6 with either DiI (D), or retroviral GFP (E), or pVIM (F). Notably, fanned fibers of PAX6+ BPs/bRGs were observed when DiI labeling was applied at the pia (D). (F) High-magnification image of a PAX6+/pVIM+ BPs/bRG, indicated by a red arrow. (G) Statistical analysis comparing number of PAX6+/pVIM+ BPs/bRGs with basal processes in the SVZ/IZ of the indicated embryos. (H, I) IHC analysis shows the expression of the human-enriched bRG markers, PTPRZ1 (H), and TNC (I), in PAX6+/TBR2- cells (arrows) of TSA-treated BAF155cKO cortex at E16.5 (see also fig. S8 for E18.5 cortex). Values are presented as means ± SEMs (* p

Techniques Used: Immunohistochemistry, Labeling, Expressing

Augmented level of H3K9ac by HDAC inhibition and overexpression of KAT2A increase basal progenitor proliferation and can induce cortical folding. (A) Staining of RG fibers (NESTIN and RC2) in Veh-treated control, TSA-treated-control and TSA-treated BAF155cKO cortex revealed an increase and divergence of radial fibers at basal sides upon TSA treatment (radial processes indicated by white-dashed lines on confocal images). (B) Cortical tissue was processed for TBR1 immunostaining. Examples with different forms of cortical folding were shown from rostral cortex to caudal cortex at E17.5. The mildly-folded and intensively-folded cortices were usually found in TSA - treated WT embryos and TSA - treated BAF155cKO embryos, respectively. (C) The folding in length was measured from the pial surface to the end of the innervated point outlined by white lines. Cortical folding phenotypes were scored as a mild folding being shorter than 200µm in length (leftmost image in B) and intensive folding with a gyrus deeper than 200µm from the surface or with more than two gyri (right images). (D) Overview of cortical phenotypes observed after TSA or Veh-treatment. In total, nine Veh-treated control, nine TSA-treated control and nine TSA-treated BAF155cKO brains at E17.5, six brains at E16.5 and six brains at E18.5/P0 of TSA-treated BAF155cKO mutants were used to examine folding phenotypes. Six coronal sections per brain covering the rostral, middle-level and caudal cortex were processed for TBR1 IHC. Cortex was scored as “mild folding” or “intensive folding”, if at least two sections met the above criteria. (E/F) Staining of ZO1 and α-Catenin in treated control (E), and BAF155cKO cortex (F) revealed an intact VZ basement membrane upon TSA treatment. (G-H) E13.5 cortices of WT or BAF155cKO embryos were electroporated with eGFP or KAT2A-ires-eGFP, followed by quantification of TBR2+/KI67+ proliferating BPs/IPs (G) and PAX6+/KI67+ proliferating BPs/bRGs (H) at E15.5. Statistical analyses revealed that the overexpression of KAT2A led to increase the proliferation of BPs. Scale bars = 100 μm.
Figure Legend Snippet: Augmented level of H3K9ac by HDAC inhibition and overexpression of KAT2A increase basal progenitor proliferation and can induce cortical folding. (A) Staining of RG fibers (NESTIN and RC2) in Veh-treated control, TSA-treated-control and TSA-treated BAF155cKO cortex revealed an increase and divergence of radial fibers at basal sides upon TSA treatment (radial processes indicated by white-dashed lines on confocal images). (B) Cortical tissue was processed for TBR1 immunostaining. Examples with different forms of cortical folding were shown from rostral cortex to caudal cortex at E17.5. The mildly-folded and intensively-folded cortices were usually found in TSA - treated WT embryos and TSA - treated BAF155cKO embryos, respectively. (C) The folding in length was measured from the pial surface to the end of the innervated point outlined by white lines. Cortical folding phenotypes were scored as a mild folding being shorter than 200µm in length (leftmost image in B) and intensive folding with a gyrus deeper than 200µm from the surface or with more than two gyri (right images). (D) Overview of cortical phenotypes observed after TSA or Veh-treatment. In total, nine Veh-treated control, nine TSA-treated control and nine TSA-treated BAF155cKO brains at E17.5, six brains at E16.5 and six brains at E18.5/P0 of TSA-treated BAF155cKO mutants were used to examine folding phenotypes. Six coronal sections per brain covering the rostral, middle-level and caudal cortex were processed for TBR1 IHC. Cortex was scored as “mild folding” or “intensive folding”, if at least two sections met the above criteria. (E/F) Staining of ZO1 and α-Catenin in treated control (E), and BAF155cKO cortex (F) revealed an intact VZ basement membrane upon TSA treatment. (G-H) E13.5 cortices of WT or BAF155cKO embryos were electroporated with eGFP or KAT2A-ires-eGFP, followed by quantification of TBR2+/KI67+ proliferating BPs/IPs (G) and PAX6+/KI67+ proliferating BPs/bRGs (H) at E15.5. Statistical analyses revealed that the overexpression of KAT2A led to increase the proliferation of BPs. Scale bars = 100 μm.

Techniques Used: Inhibition, Over Expression, Staining, Immunostaining, Immunohistochemistry

HDAC inhibition causes upregulation of bRG-enriched genes in developing mouse cortex. (A) qPCR analysis was used to confirm the upregulated expression of human-enriched bRG markers in TSA-treated cortex as compared to vehicle-treated control. (B-E) Among bRG-enriched genes, expression of TNC and PTPRZ1 have been well characterized in the developing cortex of both human and mouse (Pollen et al., 2015). TNC and PTPRZ1 antibodies labeled subsets of APs and BPs/bRGs in the entire human cortex, whereas in mouse their expression was detected only in a sub-population of PAX6+ APs in the lateral cortex (lCx, B) (Pollen et al., 2015). The increased expression of TNC (B/C) and PTPRZ1 (D) in TSA-treated WT and BAF155cKO cortex at E16.5 and E18.5 was revealed by immunolabeling (B-D) and quantification (E). Notably, the expression of these human bRG markers PTPRZ1 and TNC was found in PAX6+ APs in VZ of dorsal cortex (dCX, white filled arrows in B) and PAX6+ bRGs (white filled arrows in C, D) in TSA-treated cortex. Uppers images show the expression in arrow-pointed cells in lower pictures at higher magnification. Values are presented as mean ± SEM (*P
Figure Legend Snippet: HDAC inhibition causes upregulation of bRG-enriched genes in developing mouse cortex. (A) qPCR analysis was used to confirm the upregulated expression of human-enriched bRG markers in TSA-treated cortex as compared to vehicle-treated control. (B-E) Among bRG-enriched genes, expression of TNC and PTPRZ1 have been well characterized in the developing cortex of both human and mouse (Pollen et al., 2015). TNC and PTPRZ1 antibodies labeled subsets of APs and BPs/bRGs in the entire human cortex, whereas in mouse their expression was detected only in a sub-population of PAX6+ APs in the lateral cortex (lCx, B) (Pollen et al., 2015). The increased expression of TNC (B/C) and PTPRZ1 (D) in TSA-treated WT and BAF155cKO cortex at E16.5 and E18.5 was revealed by immunolabeling (B-D) and quantification (E). Notably, the expression of these human bRG markers PTPRZ1 and TNC was found in PAX6+ APs in VZ of dorsal cortex (dCX, white filled arrows in B) and PAX6+ bRGs (white filled arrows in C, D) in TSA-treated cortex. Uppers images show the expression in arrow-pointed cells in lower pictures at higher magnification. Values are presented as mean ± SEM (*P

Techniques Used: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Labeling, Immunolabeling

H3 acetylation controls the proliferation of BPs by activating TRNP1 expression (A, B) Western blot analysis of protein extracts from TBR2+ BPs obtained from mouse and human cortices at the indicated stages, as assessed with antibodies against TRNP1 (red), Tubulin, and GAPDH (green, as loading controls). (B) Relative levels of TRNP1 protein are presented in the diagram. Little to no TRNP1 protein is expressed in mouse BPs, whereas this expression is relatively high in human BPs. (C) ChIP-qPCR comparing the H3K9ac levels at the TRNP1 promoters in mouse and human TBR2+ BPs. (D) Schematic overview of the CRISPR/dCas9-based deposition of H3K9ac at the TRNP1 promoter, which was used to activate its expression. (E) Mouse E14.5 dorsolateral cortex was in utero electroporated with a gTRNP1-dCas9-KAT2A-T2A-eGFP plasmid (gTRNP1) or gControl-dCas9-KAT2A-T2A-eGFP plasmid (gControl), and IHC analysis of GFP, TBR2, pHH3, or BrdU was performed at E16.5. Images represent triple optical sections. White box indicates areas shown at higher magnification and red arrows point to examples of cells that are immunoreactive for GFP, TBR2, pHH3, or BrdU. (F) Descriptive scheme of the rescue experiment, in which mouse cortex was treated with the HDAC inhibitor, TSA, and gTRNP1 or shControl constructs at the indicated stages. (G) Triple IHC analysis for the markers listed in panel E. (H) Statistical quantification of the results shown in panels E and G is shown. Values are presented as mean ± SEM (* p
Figure Legend Snippet: H3 acetylation controls the proliferation of BPs by activating TRNP1 expression (A, B) Western blot analysis of protein extracts from TBR2+ BPs obtained from mouse and human cortices at the indicated stages, as assessed with antibodies against TRNP1 (red), Tubulin, and GAPDH (green, as loading controls). (B) Relative levels of TRNP1 protein are presented in the diagram. Little to no TRNP1 protein is expressed in mouse BPs, whereas this expression is relatively high in human BPs. (C) ChIP-qPCR comparing the H3K9ac levels at the TRNP1 promoters in mouse and human TBR2+ BPs. (D) Schematic overview of the CRISPR/dCas9-based deposition of H3K9ac at the TRNP1 promoter, which was used to activate its expression. (E) Mouse E14.5 dorsolateral cortex was in utero electroporated with a gTRNP1-dCas9-KAT2A-T2A-eGFP plasmid (gTRNP1) or gControl-dCas9-KAT2A-T2A-eGFP plasmid (gControl), and IHC analysis of GFP, TBR2, pHH3, or BrdU was performed at E16.5. Images represent triple optical sections. White box indicates areas shown at higher magnification and red arrows point to examples of cells that are immunoreactive for GFP, TBR2, pHH3, or BrdU. (F) Descriptive scheme of the rescue experiment, in which mouse cortex was treated with the HDAC inhibitor, TSA, and gTRNP1 or shControl constructs at the indicated stages. (G) Triple IHC analysis for the markers listed in panel E. (H) Statistical quantification of the results shown in panels E and G is shown. Values are presented as mean ± SEM (* p

Techniques Used: Expressing, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, CRISPR, In Utero, Plasmid Preparation, Immunohistochemistry, Construct

Elevated H3ac levels promote cortical expansion and folding (A) Dorsal-view images of brains from control and BAF155cKO mice with and without TSA treatment (B) and quantification of their cortical surfaces at P0 (C, see also Supplemental methods). (C-F) IHC (C) and quantitation (D-F) of the number of neuronal subsets labeled by the neuronal markers, SATB2, CTIP2, TBR1, and NeuN, in control and BAF155cKO cortices with and without HDAC inhibitor treatment at E17.5. Elevated H3 acetylation increases neurogenesis and cortical thickness and induces gyrification (indicated by arrows). (G) Serial coronal sections at E17.5 marked by CTIP2 and SATB2 expression showing the folded cortex in different cortical areas. (H) Images of GFP, SATB2, and CTIP2 immunofluorescence of a coronal section of an E17.5 cortical hemisphere from a mouse embryo that had been in utero electroporated at E12.5 with KAT2A-ires-eGFP expression plasmids. Lower images show higher-power magnification of a sulcus-like structure, which is indicated by white arrows. (I) Hypothetical model proposing how the changes in H3K9ac levels in evolution and in mouse models with epigenetic manipulation affect the H3K9ac level at the TRNP1 promoter, the expression of TRNP1, the proliferation of BPs, and the expansion and folding of the cortex. Symbols: +, +++, and ++++ indicate weak, moderate, and strong relative expression levels, respectively (Photo Credit: Dr. Tran Tuoc, Ruhr University of Bochum). Abbreviations: VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; APs, apical progenitors; BPs, basal progenitors; Ac, H3K9ac. Values are presented as mean ± SEM (*p
Figure Legend Snippet: Elevated H3ac levels promote cortical expansion and folding (A) Dorsal-view images of brains from control and BAF155cKO mice with and without TSA treatment (B) and quantification of their cortical surfaces at P0 (C, see also Supplemental methods). (C-F) IHC (C) and quantitation (D-F) of the number of neuronal subsets labeled by the neuronal markers, SATB2, CTIP2, TBR1, and NeuN, in control and BAF155cKO cortices with and without HDAC inhibitor treatment at E17.5. Elevated H3 acetylation increases neurogenesis and cortical thickness and induces gyrification (indicated by arrows). (G) Serial coronal sections at E17.5 marked by CTIP2 and SATB2 expression showing the folded cortex in different cortical areas. (H) Images of GFP, SATB2, and CTIP2 immunofluorescence of a coronal section of an E17.5 cortical hemisphere from a mouse embryo that had been in utero electroporated at E12.5 with KAT2A-ires-eGFP expression plasmids. Lower images show higher-power magnification of a sulcus-like structure, which is indicated by white arrows. (I) Hypothetical model proposing how the changes in H3K9ac levels in evolution and in mouse models with epigenetic manipulation affect the H3K9ac level at the TRNP1 promoter, the expression of TRNP1, the proliferation of BPs, and the expansion and folding of the cortex. Symbols: +, +++, and ++++ indicate weak, moderate, and strong relative expression levels, respectively (Photo Credit: Dr. Tran Tuoc, Ruhr University of Bochum). Abbreviations: VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; APs, apical progenitors; BPs, basal progenitors; Ac, H3K9ac. Values are presented as mean ± SEM (*p

Techniques Used: Mouse Assay, Immunohistochemistry, Quantitation Assay, Labeling, Expressing, Immunofluorescence, In Utero

5) Product Images from "Trichostatin A Suppresses EGFR Expression through Induction of MicroRNA-7 in an HDAC-Independent Manner in Lapatinib-Treated Cells"

Article Title: Trichostatin A Suppresses EGFR Expression through Induction of MicroRNA-7 in an HDAC-Independent Manner in Lapatinib-Treated Cells

Journal: BioMed Research International

doi: 10.1155/2014/168949

Silence of HDACs did not affect EGFR expression in lapatinib-treated MDA-MB-231 cells. (a)–(c) 231/Lap#6 cells were transfected with control siRNA or specific siRNA against HDAC3 ((a) and (c)) or HDAC7 ((b) and (c)) for 3 days. Total protein lysates prepared from these cells were subjected to Western blot analysis with indicated antibodies ((a) and (c)). Total RNA extracted from these cells was subjected to RT-qPCR. The relative mRNA levels of EGFR, HDAC3, and HDAC7 were normalized to GAPDH expression. (d) Total lysates of 231/Lap# cells transfected with myc-HDAC3 or myc-HDAC7 were subjected to immunoprecipitation with anti-myc antibody. The HDAC activities in the immunoprecipitates were measured in the HDAC activity assays. (e) and (f) 231/Lap#6 cells were transfected with increasing doses of myc-HDAC3 or myc-HDAC7 followed by treatment with TSA. Total lysates were prepared and subjected to Western blot.
Figure Legend Snippet: Silence of HDACs did not affect EGFR expression in lapatinib-treated MDA-MB-231 cells. (a)–(c) 231/Lap#6 cells were transfected with control siRNA or specific siRNA against HDAC3 ((a) and (c)) or HDAC7 ((b) and (c)) for 3 days. Total protein lysates prepared from these cells were subjected to Western blot analysis with indicated antibodies ((a) and (c)). Total RNA extracted from these cells was subjected to RT-qPCR. The relative mRNA levels of EGFR, HDAC3, and HDAC7 were normalized to GAPDH expression. (d) Total lysates of 231/Lap# cells transfected with myc-HDAC3 or myc-HDAC7 were subjected to immunoprecipitation with anti-myc antibody. The HDAC activities in the immunoprecipitates were measured in the HDAC activity assays. (e) and (f) 231/Lap#6 cells were transfected with increasing doses of myc-HDAC3 or myc-HDAC7 followed by treatment with TSA. Total lysates were prepared and subjected to Western blot.

Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Activity Assay

TSA but not SAHA suppressed lapatinib-induced EGFR expression. (a) Nuclear extract of HeLa cells was added with 1 mM TSA or SAHA for 30 min and then subjected to HDAC activity assays. (b) and (c) MDA-MB-231 and 231/Lap#6 cells were treated with indicated concentration of TSA (b) or SAHA (c) for 24 hours. Total lysates were prepared and subjected to Western blot analysis with indicated antibodies. (d) 231/Lap#6 cells were treated with 1 μ M TSA or 5 μ M SAHA for 8, 16, or 24 hours. Total lysates extracted from these cells were subjected to Western blot analysis with anti-EGFR and anti-actin antibodies. (e) 231/Lap#6 cells were treated with 1 μ M TSA or 5 μ M SAHA for 24 hours. Total RNA extracted from these cells was subjected to RT-qPCR with EGFR-specific primers. The induction of EGFR mRNA was normalized to GAPDH expression.
Figure Legend Snippet: TSA but not SAHA suppressed lapatinib-induced EGFR expression. (a) Nuclear extract of HeLa cells was added with 1 mM TSA or SAHA for 30 min and then subjected to HDAC activity assays. (b) and (c) MDA-MB-231 and 231/Lap#6 cells were treated with indicated concentration of TSA (b) or SAHA (c) for 24 hours. Total lysates were prepared and subjected to Western blot analysis with indicated antibodies. (d) 231/Lap#6 cells were treated with 1 μ M TSA or 5 μ M SAHA for 8, 16, or 24 hours. Total lysates extracted from these cells were subjected to Western blot analysis with anti-EGFR and anti-actin antibodies. (e) 231/Lap#6 cells were treated with 1 μ M TSA or 5 μ M SAHA for 24 hours. Total RNA extracted from these cells was subjected to RT-qPCR with EGFR-specific primers. The induction of EGFR mRNA was normalized to GAPDH expression.

Techniques Used: Expressing, Activity Assay, Multiple Displacement Amplification, Concentration Assay, Western Blot, Quantitative RT-PCR

6) Product Images from "Histone Deacetylases 9 and 10 Are Required for Homologous Recombination *"

Article Title: Histone Deacetylases 9 and 10 Are Required for Homologous Recombination *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.C110.194233

HDAC9 depletion sensitizes cells to mitomycin C. A , clonogenic survival on exposure to MMC was assessed on treatment of HeLa-DR cells with the HDAC inhibitors: TSA (300 n m ); apicidin (10 μ m ); VPA (2 m m ); and NaB (10 m m ). The cells were pretreated
Figure Legend Snippet: HDAC9 depletion sensitizes cells to mitomycin C. A , clonogenic survival on exposure to MMC was assessed on treatment of HeLa-DR cells with the HDAC inhibitors: TSA (300 n m ); apicidin (10 μ m ); VPA (2 m m ); and NaB (10 m m ). The cells were pretreated

Techniques Used:

7) Product Images from "Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension"

Article Title: Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2016.08.003

Inhibition of histone deacetylase (HDAC) suppresses Nox2 activity in immune cells. (A-B) Neutrophil-differentiated, HL-60 cells were treated with vehicle (DMSO) or different doses of the HDAC inhibitor, scriptaid, for long-term (24 h) or short term (30
Figure Legend Snippet: Inhibition of histone deacetylase (HDAC) suppresses Nox2 activity in immune cells. (A-B) Neutrophil-differentiated, HL-60 cells were treated with vehicle (DMSO) or different doses of the HDAC inhibitor, scriptaid, for long-term (24 h) or short term (30

Techniques Used: Inhibition, Histone Deacetylase Assay, Activity Assay

Structurally distinct HDAC inhibitors decrease Nox2 protein expression. (A-C) HL-60 cells, macrophages, and THP-1 cells were treated with vehicle (DMSO) or different inhibitors of HDACs (scriptaid, SAHA, TSA or VPA) for 24 h, and cell lysates were immunoblotted
Figure Legend Snippet: Structurally distinct HDAC inhibitors decrease Nox2 protein expression. (A-C) HL-60 cells, macrophages, and THP-1 cells were treated with vehicle (DMSO) or different inhibitors of HDACs (scriptaid, SAHA, TSA or VPA) for 24 h, and cell lysates were immunoblotted

Techniques Used: Expressing

HDAC inhibition downregulates expression of Nox 1, 2, 4 and 5 mRNA in immune, vascular and cancer cells. (A-C) HL-60 cells, and WT and Nox2−/− macrophages, were treated with or without HDAC inhibitors (scriptaid or SAHA) for 24 h, and
Figure Legend Snippet: HDAC inhibition downregulates expression of Nox 1, 2, 4 and 5 mRNA in immune, vascular and cancer cells. (A-C) HL-60 cells, and WT and Nox2−/− macrophages, were treated with or without HDAC inhibitors (scriptaid or SAHA) for 24 h, and

Techniques Used: Inhibition, Expressing

8) Product Images from "Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration"

Article Title: Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201100180

Impairment of human PANK activity in vitro results in decreased levels of tubulin and histone acetylation, an effect reverted by pantethine and by HDAC inhibitors HEK293 cells were left untreated or were treated with HoPan. Control and HoPan treated cells were co-treated with pantethine or TSA. Acetyl-lysine, acetyl-tubulin, acetyl-H3 and acetyl-H4 antibodies were used to analyse acetylation levels of tubulin and histones. Histone H2A and tubulin were used as loading controls. As in A but human SHSY-5Y cells were used. HEK293 cells transfected with a control siRNA or with an siRNA against human PANK2 were analysed for the levels of acetylated proteins. Acetyl-lysine antibody was used. Low exposure (left panel) was shown to determine levels of histone acetylation (lowest asterisk), high exposure (right panel) was shown to determine levels of acetyl-tubulin (highest asterisk). Efficiency of RNAi was confirmed using an antibody specifically recognizing human PANK2. Tubulin was used as a loading control. Control cells and human PANK2 depleted cells were investigated for their levels of acetyl H3 and acetyl H4. Cells were additionally left untreated or were treated with pantethine or VPA. Specific antibodies were used to determine levels of histone H3 and histone H4. The efficiency of human PANK2 RNAi was determined by a specific human PANK2 antibody. H3 and GAPDH were used as loading controls. Quantification of the relative levels of acetylated histone 3 and acetylated histone 4 compared to control cells.
Figure Legend Snippet: Impairment of human PANK activity in vitro results in decreased levels of tubulin and histone acetylation, an effect reverted by pantethine and by HDAC inhibitors HEK293 cells were left untreated or were treated with HoPan. Control and HoPan treated cells were co-treated with pantethine or TSA. Acetyl-lysine, acetyl-tubulin, acetyl-H3 and acetyl-H4 antibodies were used to analyse acetylation levels of tubulin and histones. Histone H2A and tubulin were used as loading controls. As in A but human SHSY-5Y cells were used. HEK293 cells transfected with a control siRNA or with an siRNA against human PANK2 were analysed for the levels of acetylated proteins. Acetyl-lysine antibody was used. Low exposure (left panel) was shown to determine levels of histone acetylation (lowest asterisk), high exposure (right panel) was shown to determine levels of acetyl-tubulin (highest asterisk). Efficiency of RNAi was confirmed using an antibody specifically recognizing human PANK2. Tubulin was used as a loading control. Control cells and human PANK2 depleted cells were investigated for their levels of acetyl H3 and acetyl H4. Cells were additionally left untreated or were treated with pantethine or VPA. Specific antibodies were used to determine levels of histone H3 and histone H4. The efficiency of human PANK2 RNAi was determined by a specific human PANK2 antibody. H3 and GAPDH were used as loading controls. Quantification of the relative levels of acetylated histone 3 and acetylated histone 4 compared to control cells.

Techniques Used: Activity Assay, In Vitro, Transfection

9) Product Images from "Suberoyl bis-hydroxamic acid reactivates Kaposi’s sarcoma-associated herpesvirus through histone acetylation and induces apoptosis in lymphoma cells"

Article Title: Suberoyl bis-hydroxamic acid reactivates Kaposi’s sarcoma-associated herpesvirus through histone acetylation and induces apoptosis in lymphoma cells

Journal: bioRxiv

doi: 10.1101/2020.09.08.288837

SBHA induced apoptosis in PEL cells. (A) Cell viability after drug treatment for 48 hours determined by trypan blue stain. (B) Dose-response relationship between SBHA concentration and cell viability. (C, D) XTT assay after drug treatment for 48 hours. Cell proliferation ratio was compared among TPA and several HDAC inhibitors (C), and dose-response relationship between SBHA concentration and cell growth was determined (D). The average from three independent experiments is shown and error bar indicates the standard deviation. (E) IC 50 of each drug against various cell lines. KSHV and EBV status are shown to the left. (F) Flow cytometry of annexin V assay. Fluorescence of FITC-labeled annexin V and PI were measured following 24 hours exposure to various concentration of SBHA. The data from control (DMSO-treated) cells and SBHA-treated (1 mM) cells are shown. (G) Dose-response relationship between SBHA concentration and apoptosis of PEL cells determined by annexin V assay.
Figure Legend Snippet: SBHA induced apoptosis in PEL cells. (A) Cell viability after drug treatment for 48 hours determined by trypan blue stain. (B) Dose-response relationship between SBHA concentration and cell viability. (C, D) XTT assay after drug treatment for 48 hours. Cell proliferation ratio was compared among TPA and several HDAC inhibitors (C), and dose-response relationship between SBHA concentration and cell growth was determined (D). The average from three independent experiments is shown and error bar indicates the standard deviation. (E) IC 50 of each drug against various cell lines. KSHV and EBV status are shown to the left. (F) Flow cytometry of annexin V assay. Fluorescence of FITC-labeled annexin V and PI were measured following 24 hours exposure to various concentration of SBHA. The data from control (DMSO-treated) cells and SBHA-treated (1 mM) cells are shown. (G) Dose-response relationship between SBHA concentration and apoptosis of PEL cells determined by annexin V assay.

Techniques Used: Staining, Concentration Assay, XTT Assay, Standard Deviation, Flow Cytometry, Annexin V Assay, Fluorescence, Labeling

10) Product Images from "Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells"

Article Title: Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02838

HDAC inhibitor mimicked the butyrate-dependent down-regulation of IDO-1 expression in a SP1, PPARγ and AP-1 independent manner. (A) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2 mM), SAHA (5 μM), Trichostatin A (TSA 1 μM) or Valproic acid (VAP 5 mM) ± SP1 inhibitor (Mitramycin A; MitA 0.1 μM). (B) , HT-29- IDO-1 reporter cells were stimulated for 24 h with two PPARγ activators: Pioglitazone (Pio 5 μM); Rosiglitazone (Rosi, 10 μM) or the specific PPARγ inhibitor GW9662 (10 μM) ± butyrate (But 2 mM). (C) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2mM) and/or the AP1 inhibitor, SR11302 (10 μM). IDO-1 expression was measured by luciferase activity and expressed as median ± quartiles of fold change toward un-stimulated cells. Data represented at least three independent experiments. P -value: *** P
Figure Legend Snippet: HDAC inhibitor mimicked the butyrate-dependent down-regulation of IDO-1 expression in a SP1, PPARγ and AP-1 independent manner. (A) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2 mM), SAHA (5 μM), Trichostatin A (TSA 1 μM) or Valproic acid (VAP 5 mM) ± SP1 inhibitor (Mitramycin A; MitA 0.1 μM). (B) , HT-29- IDO-1 reporter cells were stimulated for 24 h with two PPARγ activators: Pioglitazone (Pio 5 μM); Rosiglitazone (Rosi, 10 μM) or the specific PPARγ inhibitor GW9662 (10 μM) ± butyrate (But 2 mM). (C) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2mM) and/or the AP1 inhibitor, SR11302 (10 μM). IDO-1 expression was measured by luciferase activity and expressed as median ± quartiles of fold change toward un-stimulated cells. Data represented at least three independent experiments. P -value: *** P

Techniques Used: Expressing, Incubation, Luciferase, Activity Assay

11) Product Images from "The Inflammatory Cytokines TWEAK and TNF? Reduce Renal Klotho Expression through NF?B"

Article Title: The Inflammatory Cytokines TWEAK and TNF? Reduce Renal Klotho Expression through NF?B

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2010101073

TWEAK- and TNFα-mediated downregulation of Klotho mRNA in cultured tubular cells requires HDAC activity. (A) The HDAC inhibitor TSA rescues TWEAK- or TNFα-induced repression of Klotho at 3 hours in MCT cells. Cells were prestimulated with
Figure Legend Snippet: TWEAK- and TNFα-mediated downregulation of Klotho mRNA in cultured tubular cells requires HDAC activity. (A) The HDAC inhibitor TSA rescues TWEAK- or TNFα-induced repression of Klotho at 3 hours in MCT cells. Cells were prestimulated with

Techniques Used: Cell Culture, Activity Assay

12) Product Images from "RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing"

Article Title: RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1134

HDAC inhibition favors exclusion of alternative exon 2 upon TPA induction of MCL1 gene. ( A ) MCL1 alternative exon 2 splicing was analyzed in TPA-stimulated serum-starved HCT116 cells treated or not with 150 nM apicidin prior to TPA induction. ( B ) Schematic representation of the amplicons generated by real-time PCR analyses of ChIP DNA. ( C ) ChIP assays were performed on DSP- and formaldehyde-crosslinked mononucleosomes prepared from TPA-stimulated serum-starved HCT116 cells treated or not with 150 nM apicidin for 30 min prior to TPA induction.
Figure Legend Snippet: HDAC inhibition favors exclusion of alternative exon 2 upon TPA induction of MCL1 gene. ( A ) MCL1 alternative exon 2 splicing was analyzed in TPA-stimulated serum-starved HCT116 cells treated or not with 150 nM apicidin prior to TPA induction. ( B ) Schematic representation of the amplicons generated by real-time PCR analyses of ChIP DNA. ( C ) ChIP assays were performed on DSP- and formaldehyde-crosslinked mononucleosomes prepared from TPA-stimulated serum-starved HCT116 cells treated or not with 150 nM apicidin for 30 min prior to TPA induction.

Techniques Used: Inhibition, Generated, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

SRSF1, HDAC1 and HDAC2 regulate alternative splicing of MCL1 RNA. ( A ) Schematic representation of MCL1 alternative splicing. Arrows above and below map represent primers used in reverse transcription PCR (RT–PCR) assays. MCL1 mature mRNAs were visualized and quantified on denaturing polyacrylamide gels after 32 P-labeling RT–PCR, in HCT116 cycling cells following ( B ) SRSF1 knockdown, ( C ) HDAC inhibitor (250 nM TSA or 150 nM apicidin) 2 h treatment or ( D ) HDAC1, HDAC2 or HDAC1/2 knockdown. Percentage of exon 2 exclusion was calculated by measuring the signal intensity of the spliced transcript ( MCL1S ) over the total transcripts ( MCL1S + MCL1L ). The average of at least three experiments is shown including SD, ** indicates P ≤ 0.01 and * P ≤ 0.05 of the t -test.
Figure Legend Snippet: SRSF1, HDAC1 and HDAC2 regulate alternative splicing of MCL1 RNA. ( A ) Schematic representation of MCL1 alternative splicing. Arrows above and below map represent primers used in reverse transcription PCR (RT–PCR) assays. MCL1 mature mRNAs were visualized and quantified on denaturing polyacrylamide gels after 32 P-labeling RT–PCR, in HCT116 cycling cells following ( B ) SRSF1 knockdown, ( C ) HDAC inhibitor (250 nM TSA or 150 nM apicidin) 2 h treatment or ( D ) HDAC1, HDAC2 or HDAC1/2 knockdown. Percentage of exon 2 exclusion was calculated by measuring the signal intensity of the spliced transcript ( MCL1S ) over the total transcripts ( MCL1S + MCL1L ). The average of at least three experiments is shown including SD, ** indicates P ≤ 0.01 and * P ≤ 0.05 of the t -test.

Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Labeling

13) Product Images from "Histone deacetylase inhibitors prevent oxidative neuronal death independent of expanded polyglutamine repeats via an Sp1-dependent pathway"

Article Title: Histone deacetylase inhibitors prevent oxidative neuronal death independent of expanded polyglutamine repeats via an Sp1-dependent pathway

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

doi: 10.1073/pnas.0737363100

HDAC inhibitors augment Sp1 acetylation, Sp1 DNA binding activity, and Sp1-dependent reporter gene expression in cortical neuronal cultures. ( A ) Sp1 acetylation levels in cortical neurons treated with the prototypic HDAC inhibitor TSA as determined by immunoprecipitation with an Sp1 Ab followed by immunoblotting with acetyl lysine Ab (Ac-Sp1) or Sp1 Ab alone (Sp1). Note that levels of Sp1 do not change with increasing concentrations of TSA. ( B ) TSA enhances binding of Sp1 and Sp3 to a canonical Sp1 DNA binding site. The electrophoretic mobility-shift assay was performed by using nuclear extracts from cortical neurons treated with and without TSA (100 ng/ml) for 60 min. The presence of Sp1 and Sp3 in each of the induced complexes was verified by supershift analysis. During this short period of TSA exposure, Sp1 or Sp3 protein levels did not change. ( C ) Sp1-dependent luciferase activity in control and TSA (100 ng/ml)-treated cortical neurons (gray bars). Note that luciferase activity does not change in the presence of TSA when the Sp1 response element has been mutated (black bars).
Figure Legend Snippet: HDAC inhibitors augment Sp1 acetylation, Sp1 DNA binding activity, and Sp1-dependent reporter gene expression in cortical neuronal cultures. ( A ) Sp1 acetylation levels in cortical neurons treated with the prototypic HDAC inhibitor TSA as determined by immunoprecipitation with an Sp1 Ab followed by immunoblotting with acetyl lysine Ab (Ac-Sp1) or Sp1 Ab alone (Sp1). Note that levels of Sp1 do not change with increasing concentrations of TSA. ( B ) TSA enhances binding of Sp1 and Sp3 to a canonical Sp1 DNA binding site. The electrophoretic mobility-shift assay was performed by using nuclear extracts from cortical neurons treated with and without TSA (100 ng/ml) for 60 min. The presence of Sp1 and Sp3 in each of the induced complexes was verified by supershift analysis. During this short period of TSA exposure, Sp1 or Sp3 protein levels did not change. ( C ) Sp1-dependent luciferase activity in control and TSA (100 ng/ml)-treated cortical neurons (gray bars). Note that luciferase activity does not change in the presence of TSA when the Sp1 response element has been mutated (black bars).

Techniques Used: Binding Assay, Activity Assay, Expressing, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Luciferase

HDAC inhibitors inhibit neuronal death induced by glutathione-depletion-induced oxidative stress. ( A ) TSA inhibits HCA-induced apoptosis in a concentration-dependent manner. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. Lactate dehydrogenase (LDH) release assay and TUNEL were performed in parallel to verify that MTT changes reflect changes in viability. Each point is the mean ± SD of three to five independent experiments. ( B ) Phase-contrast microscopy of cortical neurons: ( a ) control; ( b ) 1 mM HCA; ( c ) 100 ng/ml TSA (note the change in the morphology of the cell bodies); and ( d ) 100 ng/ml TSA plus 1 mM HCA (note how TSA-treated neurons maintain their cell body and neurite morphology in the presence of 1 mM HCA). (Magnifications: ×200.) ( C ) Structurally distinct HDAC inhibitors, butyrate (5 mM) and SAHA (5 μM), also inhibit HCA-induced death. Cell viability was measured by using the MTT reduction, LDH release, or TUNEL as described in Experimental Methods . All methods gave quantitatively similar results.
Figure Legend Snippet: HDAC inhibitors inhibit neuronal death induced by glutathione-depletion-induced oxidative stress. ( A ) TSA inhibits HCA-induced apoptosis in a concentration-dependent manner. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. Lactate dehydrogenase (LDH) release assay and TUNEL were performed in parallel to verify that MTT changes reflect changes in viability. Each point is the mean ± SD of three to five independent experiments. ( B ) Phase-contrast microscopy of cortical neurons: ( a ) control; ( b ) 1 mM HCA; ( c ) 100 ng/ml TSA (note the change in the morphology of the cell bodies); and ( d ) 100 ng/ml TSA plus 1 mM HCA (note how TSA-treated neurons maintain their cell body and neurite morphology in the presence of 1 mM HCA). (Magnifications: ×200.) ( C ) Structurally distinct HDAC inhibitors, butyrate (5 mM) and SAHA (5 μM), also inhibit HCA-induced death. Cell viability was measured by using the MTT reduction, LDH release, or TUNEL as described in Experimental Methods . All methods gave quantitatively similar results.

Techniques Used: High Content Screening, Concentration Assay, MTT Assay, Lactate Dehydrogenase Assay, TUNEL Assay, Microscopy

Sp1 is necessary for the protective effects of HDAC inhibitors. ( A ) Sp1 AS ODNs, but not MM ODNs, deplete Sp1 protein levels in cortical neurons. Sp1 ODNs do not alter the levels of α-tubulin. ( B ) Sp1 AS ODNs reverse TSA-induced prevention of HCA-induced death; MM ODNs do not. Results are mean ± SE for three separate experiments.
Figure Legend Snippet: Sp1 is necessary for the protective effects of HDAC inhibitors. ( A ) Sp1 AS ODNs, but not MM ODNs, deplete Sp1 protein levels in cortical neurons. Sp1 ODNs do not alter the levels of α-tubulin. ( B ) Sp1 AS ODNs reverse TSA-induced prevention of HCA-induced death; MM ODNs do not. Results are mean ± SE for three separate experiments.

Techniques Used: High Content Screening

14) Product Images from "The interplay between histone deacetylases and c-Myc in the transcriptional suppression of HPP1 in colon cancer"

Article Title: The interplay between histone deacetylases and c-Myc in the transcriptional suppression of HPP1 in colon cancer

Journal: Cancer Biology & Therapy

doi: 10.4161/cbt.29500

Figure 1. Expression of HPP1 in colon cancer cell lines. DLD-1, HCT116, MOCK (HCT116 transfected with empty vector control), and HPP1 (HCT116 transfected with full-length HPP1 ) by quantitative RT-PCR ( A ) and western blot ( B ): HPP1 is re-expressed in HCT116 ( C ) and DLD-1 ( D ) cells treated with HDAC inhibitors (SB 5 mM, SAHA 5 μM, TSA 200 nM, and VPA 2 mM) when compared with vehicle alone (control) at different time points. The re-expression of HPP1 at 24 h increased significantly by qRT-PCR in response to HDAC inhibitors ( E ). A similar trend in protein expression was also observed by western blot analyses ( F ).
Figure Legend Snippet: Figure 1. Expression of HPP1 in colon cancer cell lines. DLD-1, HCT116, MOCK (HCT116 transfected with empty vector control), and HPP1 (HCT116 transfected with full-length HPP1 ) by quantitative RT-PCR ( A ) and western blot ( B ): HPP1 is re-expressed in HCT116 ( C ) and DLD-1 ( D ) cells treated with HDAC inhibitors (SB 5 mM, SAHA 5 μM, TSA 200 nM, and VPA 2 mM) when compared with vehicle alone (control) at different time points. The re-expression of HPP1 at 24 h increased significantly by qRT-PCR in response to HDAC inhibitors ( E ). A similar trend in protein expression was also observed by western blot analyses ( F ).

Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

15) Product Images from "HDAC1/2-Mediated Regulation of JNK and ERK Phosphorylation in Bovine Mammary Epithelial Cells (MAC-Ts) in response to TNFα"

Article Title: HDAC1/2-Mediated Regulation of JNK and ERK Phosphorylation in Bovine Mammary Epithelial Cells (MAC-Ts) in response to TNFα

Journal: Journal of cellular physiology

doi: 10.1002/jcp.27265

Class I HDACs regulate MAC-T inflammatory gene expression in response to TNFα. MAC-T cells under basal (10% FBS, 10 mg/ml insulin) conditions were treated with the class I selective HDAC inhibitor MGCD for 24 hrs prior to TNFα stimulation (300 pM) and RNA isolated at 1hr post-TNFα stimulation. A) Heatmap generated from a quantitative polymerase chain reaction (qPCR) array was used to examine inflammatory gene expression. B) Real-time qPCR was used to validate CCL26, TNFα, CSF2 and LTB gene expression from mRNA identified in the array. C) In addition, MAC-T cells were pretreated with an independent class I selective HDAC inhibitor apicidin (Api; 1 μM) or an HDAC1/2-specific inhibitor (Romidepsin, Rom 1 μM) for 24 hrs prior to TNFα stimulation (300 pM). RNA was isolated 1hr post-TNFα stimulation and qPCR used to examine CCL26, TNFα, CSF2 and LTB gene expression. One-way ANOVA with Tukey’s post-hoc analysis was used to assess statistical significance when p
Figure Legend Snippet: Class I HDACs regulate MAC-T inflammatory gene expression in response to TNFα. MAC-T cells under basal (10% FBS, 10 mg/ml insulin) conditions were treated with the class I selective HDAC inhibitor MGCD for 24 hrs prior to TNFα stimulation (300 pM) and RNA isolated at 1hr post-TNFα stimulation. A) Heatmap generated from a quantitative polymerase chain reaction (qPCR) array was used to examine inflammatory gene expression. B) Real-time qPCR was used to validate CCL26, TNFα, CSF2 and LTB gene expression from mRNA identified in the array. C) In addition, MAC-T cells were pretreated with an independent class I selective HDAC inhibitor apicidin (Api; 1 μM) or an HDAC1/2-specific inhibitor (Romidepsin, Rom 1 μM) for 24 hrs prior to TNFα stimulation (300 pM). RNA was isolated 1hr post-TNFα stimulation and qPCR used to examine CCL26, TNFα, CSF2 and LTB gene expression. One-way ANOVA with Tukey’s post-hoc analysis was used to assess statistical significance when p

Techniques Used: Expressing, Isolation, Generated, Real-time Polymerase Chain Reaction

Class I HDACs regulate JNK and ERK phosphorylation in MAC-T cells in response to TNFα. MAC-T cells under basal (10% FBS, 10 mg/ml insulin) conditions were A) treated with trichostatin A (TSA) for 24 hrs prior to TNFα stimulation (300pM). Cell lysates were harvested at 15 min post-TNFα stimulation and immunoblotted for phospho-JNK, phospho-p38, phosphor-ERK, total-JNK, total-ERK and total-p38. B) MAC-T cells were treated with class selective HDAC inhibitors MGCD0103 (MGCD; class I), DPAH (Class IIa) and Tubastating A (TubA; class IIb) prior to TNFα (300 pM) stimulation. Cell lysates were collected 15 min post-TNFα stimulation and immunoblotted for phospho-JNK, phospho-p38, phosphor-ERK as well as total-JNK, total-ERK and total-p38. C) MAC-T cells were treated with class I selective HDAC inhibitors MGCD and apicidin (Api) 24 hrs prior to TNFα (300 pM) stimulation. Cells lysates were harvested 15 min post-TNFα stimulation and immunoblotted as above. D) Protein was harvested from MAC-T cells treated with basal (10% FBS, 10 mg/ml insulin) media. Protein lysate (30 μg) was subsequently incubated with vehicle (DMSO), TSA, MGCD, and apicidin for 2 hrs prior to addition of class-specific HDAC substrates. Developer solution was added for an additional 20 minutes and fluorescent activity measured via BioTek plate reader. One-way ANOVA with Dunnett’s post-hoc analysis was used to assess statistical significance when p
Figure Legend Snippet: Class I HDACs regulate JNK and ERK phosphorylation in MAC-T cells in response to TNFα. MAC-T cells under basal (10% FBS, 10 mg/ml insulin) conditions were A) treated with trichostatin A (TSA) for 24 hrs prior to TNFα stimulation (300pM). Cell lysates were harvested at 15 min post-TNFα stimulation and immunoblotted for phospho-JNK, phospho-p38, phosphor-ERK, total-JNK, total-ERK and total-p38. B) MAC-T cells were treated with class selective HDAC inhibitors MGCD0103 (MGCD; class I), DPAH (Class IIa) and Tubastating A (TubA; class IIb) prior to TNFα (300 pM) stimulation. Cell lysates were collected 15 min post-TNFα stimulation and immunoblotted for phospho-JNK, phospho-p38, phosphor-ERK as well as total-JNK, total-ERK and total-p38. C) MAC-T cells were treated with class I selective HDAC inhibitors MGCD and apicidin (Api) 24 hrs prior to TNFα (300 pM) stimulation. Cells lysates were harvested 15 min post-TNFα stimulation and immunoblotted as above. D) Protein was harvested from MAC-T cells treated with basal (10% FBS, 10 mg/ml insulin) media. Protein lysate (30 μg) was subsequently incubated with vehicle (DMSO), TSA, MGCD, and apicidin for 2 hrs prior to addition of class-specific HDAC substrates. Developer solution was added for an additional 20 minutes and fluorescent activity measured via BioTek plate reader. One-way ANOVA with Dunnett’s post-hoc analysis was used to assess statistical significance when p

Techniques Used: Incubation, Activity Assay

16) Product Images from "Synergistic enhancement of NK cell-mediated cytotoxicity by combination of histone deacetylase inhibitor and ionizing radiation"

Article Title: Synergistic enhancement of NK cell-mediated cytotoxicity by combination of histone deacetylase inhibitor and ionizing radiation

Journal: Radiation Oncology (London, England)

doi: 10.1186/1748-717X-9-49

Cell surface expressions of NKG2D ligands after treating irradiated A549 cells with HDAC inhibitors. NKG2D ligand cell surface expressions were analyzed by flow cytometry using specific mAbs against NKG2D ligands. A549 cells were irradiated with 8 Gy, allowed to recover for 6 hours, and then treated with or without 250nM TSA or 125 ng/ml apicidin. The cells were then incubated for 18 hours. In the figure, filled gray represents the isotype control and filled black represents the treated group (A) . All experiments performed independently in triplicate and significant differences between the untreated control and treated cells are presented as * (p
Figure Legend Snippet: Cell surface expressions of NKG2D ligands after treating irradiated A549 cells with HDAC inhibitors. NKG2D ligand cell surface expressions were analyzed by flow cytometry using specific mAbs against NKG2D ligands. A549 cells were irradiated with 8 Gy, allowed to recover for 6 hours, and then treated with or without 250nM TSA or 125 ng/ml apicidin. The cells were then incubated for 18 hours. In the figure, filled gray represents the isotype control and filled black represents the treated group (A) . All experiments performed independently in triplicate and significant differences between the untreated control and treated cells are presented as * (p

Techniques Used: Irradiation, Flow Cytometry, Cytometry, Incubation

Increased susceptibility of A549 cells to the cytolytic activity of NK-92 cells following HDAC inhibitor and ionizing radiation co-treatment. A549 cells were not treated (open circle), irradiated with 8 Gy (filled circle), treated with 125 ng/ml apicidin or 250 nM TSA (filled square), or co-treated with after ionizing radiation plus HDAC inhibitors (apicidin or TSA; filled triangle). Cells were co-cultured with NK-92 cells at the indicated effector/target ratio. To determine the specificity of NKG2D-mediated cytolysis, NK-92 cells were pre-incubated with blocking mAb against NKG2D (open triangle). All experiments were performed in triplicate and significant differences between NK cell-mediated lyses of untreated and treated cells were accepted for P values of
Figure Legend Snippet: Increased susceptibility of A549 cells to the cytolytic activity of NK-92 cells following HDAC inhibitor and ionizing radiation co-treatment. A549 cells were not treated (open circle), irradiated with 8 Gy (filled circle), treated with 125 ng/ml apicidin or 250 nM TSA (filled square), or co-treated with after ionizing radiation plus HDAC inhibitors (apicidin or TSA; filled triangle). Cells were co-cultured with NK-92 cells at the indicated effector/target ratio. To determine the specificity of NKG2D-mediated cytolysis, NK-92 cells were pre-incubated with blocking mAb against NKG2D (open triangle). All experiments were performed in triplicate and significant differences between NK cell-mediated lyses of untreated and treated cells were accepted for P values of

Techniques Used: Activity Assay, Irradiation, Cell Culture, Incubation, Blocking Assay

Analysis of the expressions of NKG2D ligands after treating lung cancer cells with HDAC inhibitors or ionizing radiation. Multiplex RT-PCR analysis was performed to determine the mRNA expressions of NKG2D ligands. A549 and NCI-H23 cells were treated with 125 ng/ml apicidin, 2.5 μ M SAHA, or 250 nM TSA (A, B) for 12 hours. A549 cells were irradiated with the indicated doses and allowed to recover for 24 hours (C) . Amplified DNA was quantitated using Quantity-one software (Bio-rad). All experiments were performed at least three times, and representative results are shown. MICA/B: MHC class I chain-related molecules A/B, ULBP1/2/3: UL16-binding proteins 1/2/3, ACTB: -actin, RPL19: ribosomal protein19.
Figure Legend Snippet: Analysis of the expressions of NKG2D ligands after treating lung cancer cells with HDAC inhibitors or ionizing radiation. Multiplex RT-PCR analysis was performed to determine the mRNA expressions of NKG2D ligands. A549 and NCI-H23 cells were treated with 125 ng/ml apicidin, 2.5 μ M SAHA, or 250 nM TSA (A, B) for 12 hours. A549 cells were irradiated with the indicated doses and allowed to recover for 24 hours (C) . Amplified DNA was quantitated using Quantity-one software (Bio-rad). All experiments were performed at least three times, and representative results are shown. MICA/B: MHC class I chain-related molecules A/B, ULBP1/2/3: UL16-binding proteins 1/2/3, ACTB: -actin, RPL19: ribosomal protein19.

Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Irradiation, Amplification, Software, Binding Assay

17) Product Images from "Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency"

Article Title: Human endogenous retrovirus (HERV) expression is not induced by treatment with the histone deacetylase (HDAC) inhibitors in cellular models of HIV-1 latency

Journal: Retrovirology

doi: 10.1186/s12977-016-0242-4

The HDAC inhibitors promote histone acetylation and HIV-1 p24 expression. a Treatment of U1 cells with HDAC inhibitors results in increased acetylation of histone H4. The blot was probed with antibody to acetylated H4, followed by HRP-conjugated secondary antibody and enhanced chemiluminescence (ECL) detection. Subsequently, the same blots were reprobed with anti-β-actin antibody. The lanes are ( 1 ) untreated, ( 2 ) vorinostat, ( 3 ) panobinostat, ( 4 ) romidepsin, ( 5 ) prostratin and ( 6 ) PMA. b HIV-1 p24 expression is induced by vorinostat, panobinostat, romidepsin, prostratin and PMA, as measured by flow cytometry
Figure Legend Snippet: The HDAC inhibitors promote histone acetylation and HIV-1 p24 expression. a Treatment of U1 cells with HDAC inhibitors results in increased acetylation of histone H4. The blot was probed with antibody to acetylated H4, followed by HRP-conjugated secondary antibody and enhanced chemiluminescence (ECL) detection. Subsequently, the same blots were reprobed with anti-β-actin antibody. The lanes are ( 1 ) untreated, ( 2 ) vorinostat, ( 3 ) panobinostat, ( 4 ) romidepsin, ( 5 ) prostratin and ( 6 ) PMA. b HIV-1 p24 expression is induced by vorinostat, panobinostat, romidepsin, prostratin and PMA, as measured by flow cytometry

Techniques Used: Expressing, Flow Cytometry, Cytometry

The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in J-LAT-8.4 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL). The data points ( empty circles ) represent the relative fold change in expression normalised with GAPDH ( black squares show the median and orange lines show 95 % CI) for up to three replicates in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in J-LAT-8.4 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL). The data points ( empty circles ) represent the relative fold change in expression normalised with GAPDH ( black squares show the median and orange lines show 95 % CI) for up to three replicates in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

Techniques Used: Expressing

The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells following treatment with higher doses of the drugs for 5 h. The doses of the drugs used in this experiments were: vorinostat 1 μM/well, panobinostat 0.1 μM/well and PMA 0.1 μg/μL. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells following treatment with higher doses of the drugs for 5 h. The doses of the drugs used in this experiments were: vorinostat 1 μM/well, panobinostat 0.1 μM/well and PMA 0.1 μg/μL. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates in two independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

Techniques Used: Expressing

The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL) and IL-1β (10 pg/mL). The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates ( lines show 95 % CI) in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)
Figure Legend Snippet: The HDAC inhibitors panobinostat and vorinostat do not increase HERV expression in U1 cells. The HERVs analysed were: HK2 env , HK2 pol , HERV-W env (syncytin-1) and HERV-FRD env (syncytin-2). The fold change in HERV expression following drug treatment was compared to the untreated control ( lines show 95 % CI) and was calculated relative to GAPDH expression. The doses of the drugs used were vorinostat (1 μM/well), panobinostat (0.1 μM/well), PMA (0.1 μg/μL) and IL-1β (10 pg/mL). The data points represent the relative fold change in expression normalised with GAPDH ( lines show 95 % CI) for up to three replicates ( lines show 95 % CI) in four independent experiments. A significant change of expression (i.e. higher than the untreated cells) would show the 95 % CI to be higher than and not overlap the dashed horizontal line which indicates 1× relative fold change (two-sided test)

Techniques Used: Expressing

18) Product Images from "Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption"

Article Title: Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption

Journal: Arthritis Research & Therapy

doi: 10.1186/ar1702

HDAC inhibitors decrease collagenolytic and gelatinolytic activity from bovine nasal explants and block collagenase activation. Conditioned media from cartilage assays (day 14) as in Fig. 1a were assayed (a) for collagenase activity in the presence or absence of 0.67 mM APMA, an activator of procollagenases (means ± standard errors of the mean), and (b) for gelatinase activity, using gelatin zymography. APMA, aminophenylmercuric acetate; HDAC, histone deacetylase; I/O, a combination of IL-1α and OSM; MMP, matrix metalloproteinase; OSM, oncostatin M; TSA, trichostatin A.
Figure Legend Snippet: HDAC inhibitors decrease collagenolytic and gelatinolytic activity from bovine nasal explants and block collagenase activation. Conditioned media from cartilage assays (day 14) as in Fig. 1a were assayed (a) for collagenase activity in the presence or absence of 0.67 mM APMA, an activator of procollagenases (means ± standard errors of the mean), and (b) for gelatinase activity, using gelatin zymography. APMA, aminophenylmercuric acetate; HDAC, histone deacetylase; I/O, a combination of IL-1α and OSM; MMP, matrix metalloproteinase; OSM, oncostatin M; TSA, trichostatin A.

Techniques Used: Activity Assay, Blocking Assay, Activation Assay, Zymography, Histone Deacetylase Assay

19) Product Images from "Dual inhibition of AKT‐m TOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer"

Article Title: Dual inhibition of AKT‐m TOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201708478

HDAC 3 associates with AKT in plasma membrane and induces lysine 63‐linked ubiquitination of AKT LNCaP and C4‐2 cells grown in logarithmic phase were fixed and subjected to IFC. Cell nuclei were counterstained by DAPI. Arrows point to the co‐localization of AKT and HDAC3 proteins in the plasma membrane. Approximately 80% of LNCaP cells and 85% of C4‐2 cells showed the co‐localization of these two proteins on plasma membrane. Scale bars, 20 μm. C4‐2 cells were treated with IGF‐1 for 30 min and then harvested for cellular fractionation followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with empty vector (EV) or HA‐tagged wild‐type or mutant HDAC3 for 24 h followed by treatment of 10 ng/ml IGF‐1 for 30 min. Cells were harvested for Western blots with the indicated antibodies. C4‐2 cells were transfected with the indicated plasmids for 24 h and harvested for IP and Western blots with the indicated antibodies. Source data are available online for this figure.
Figure Legend Snippet: HDAC 3 associates with AKT in plasma membrane and induces lysine 63‐linked ubiquitination of AKT LNCaP and C4‐2 cells grown in logarithmic phase were fixed and subjected to IFC. Cell nuclei were counterstained by DAPI. Arrows point to the co‐localization of AKT and HDAC3 proteins in the plasma membrane. Approximately 80% of LNCaP cells and 85% of C4‐2 cells showed the co‐localization of these two proteins on plasma membrane. Scale bars, 20 μm. C4‐2 cells were treated with IGF‐1 for 30 min and then harvested for cellular fractionation followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with empty vector (EV) or HA‐tagged wild‐type or mutant HDAC3 for 24 h followed by treatment of 10 ng/ml IGF‐1 for 30 min. Cells were harvested for Western blots with the indicated antibodies. C4‐2 cells were transfected with the indicated plasmids for 24 h and harvested for IP and Western blots with the indicated antibodies. Source data are available online for this figure.

Techniques Used: Cell Fractionation, Western Blot, Transfection, Plasmid Preparation, Mutagenesis

HDAC 3 is important for growth factor‐induced AKT deacetylation and polyubiquitination 293T cells were transfected with plasmids for Flag‐HDAC3 and Myc‐AKT followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with plasmids for Myc‐AKT, HA‐Ub, and Flag‐HDAC3 (different doses) for 24 h followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with plasmids for Myc‐AKT and Flag‐HDAC3 (different doses) for 24 h followed by Western blots with the indicated antibodies. C4‐2 cells were transfected with a pool of control or HDAC3‐specific siRNAs for 48 h followed by IP and/or Western blots with the indicated antibodies. C4‐2 cells were transfected with HA‐Ub plasmids and treated with 3 μM of RGFP966 for 24 h followed by IP and/or Western blots with the indicated antibodies. C4‐2 cells were treated with 10 ng/ml of IGF‐1 for different periods of time and harvested for IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with a pool of control and HDAC3‐specific siRNAs for 48 h and then treated with IGF‐1 (H) or EGF (I) followed by Western blots for indicated proteins. Data information: The asterisk (*) indicates the specific HDAC3 protein band. Source data are available online for this figure.
Figure Legend Snippet: HDAC 3 is important for growth factor‐induced AKT deacetylation and polyubiquitination 293T cells were transfected with plasmids for Flag‐HDAC3 and Myc‐AKT followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with plasmids for Myc‐AKT, HA‐Ub, and Flag‐HDAC3 (different doses) for 24 h followed by IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with plasmids for Myc‐AKT and Flag‐HDAC3 (different doses) for 24 h followed by Western blots with the indicated antibodies. C4‐2 cells were transfected with a pool of control or HDAC3‐specific siRNAs for 48 h followed by IP and/or Western blots with the indicated antibodies. C4‐2 cells were transfected with HA‐Ub plasmids and treated with 3 μM of RGFP966 for 24 h followed by IP and/or Western blots with the indicated antibodies. C4‐2 cells were treated with 10 ng/ml of IGF‐1 for different periods of time and harvested for IP and Western blots with the indicated antibodies. C4‐2 cells were transfected with a pool of control and HDAC3‐specific siRNAs for 48 h and then treated with IGF‐1 (H) or EGF (I) followed by Western blots for indicated proteins. Data information: The asterisk (*) indicates the specific HDAC3 protein band. Source data are available online for this figure.

Techniques Used: Transfection, Western Blot

The C‐terminal region (aa 1–313) in HDAC 3 is indispensable for its cytoplasm exportation that is a prerequisite for AKT phosphorylation and AR upregulation LNCaP cells were treated with 10 ng of IGF‐1 for 30 m and harvested for immunofluorescent cytochemistry (IFC) with P‐AKT‐S473, E‐cadherin, and HDAC3. Cell nuclei were counterstained with DAPI in IFC. Scale bars, 50 μm. C4‐2 cells were transfected with the indicated plasmids, and then, cytoplasm membrane and nuclear proteins were isolated followed by IP and Western blots with the indicated antibodies. An illustration depicts functional domains of HDAC3 including the regions for nuclear export and localization sequences (modified from the following website: http://atlasgeneticsoncology.org/Genes/GC_HDAC3.html ). LNCaP cells were transfected with HA‐tagged wild‐type HDAC3 or truncated mutant (aa 1–313) for 24 h followed by IFC with anti‐HA antibody (D) or Western blots with indicated antibodies (E). Scale bars for images in (D), 50 μm. C4‐2 cells were transfected with a pool of siRNA of APLL1 and the indicated plasmids. The cells were harvested for IP and Western blots with the indicated antibodies. Source data are available online for this figure.
Figure Legend Snippet: The C‐terminal region (aa 1–313) in HDAC 3 is indispensable for its cytoplasm exportation that is a prerequisite for AKT phosphorylation and AR upregulation LNCaP cells were treated with 10 ng of IGF‐1 for 30 m and harvested for immunofluorescent cytochemistry (IFC) with P‐AKT‐S473, E‐cadherin, and HDAC3. Cell nuclei were counterstained with DAPI in IFC. Scale bars, 50 μm. C4‐2 cells were transfected with the indicated plasmids, and then, cytoplasm membrane and nuclear proteins were isolated followed by IP and Western blots with the indicated antibodies. An illustration depicts functional domains of HDAC3 including the regions for nuclear export and localization sequences (modified from the following website: http://atlasgeneticsoncology.org/Genes/GC_HDAC3.html ). LNCaP cells were transfected with HA‐tagged wild‐type HDAC3 or truncated mutant (aa 1–313) for 24 h followed by IFC with anti‐HA antibody (D) or Western blots with indicated antibodies (E). Scale bars for images in (D), 50 μm. C4‐2 cells were transfected with a pool of siRNA of APLL1 and the indicated plasmids. The cells were harvested for IP and Western blots with the indicated antibodies. Source data are available online for this figure.

Techniques Used: Transfection, Isolation, Western Blot, Functional Assay, Modification, Mutagenesis

The scaffold protein APPL 1 facilitates HDAC 3 regulation of AKT C4‐2 cell lysate was prepared for IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cell lysate was prepared for GST pull‐down assay using GST or GST‐HDAC3 recombinant proteins (stained with Coomassie blue, low panel) followed by Western blot with anti‐APPL1 antibody (upper panel). GST or GST‐HDAC3 recombinant proteins with expected molecular mass are indicated by asterisks. An illustration depicts four functional domains (BAR, PH, PTB, and PDZ) of APPL1 used for construction of GST‐APPL1 recombinant proteins. C4‐2 cell lysate was prepared for GST pull‐down assay using GST or GST‐APPL1 recombinant proteins (stained with Coomassie blue, low panel) followed by Western blot with anti‐HDAC3 antibody (upper panel). The red asterisk (*) indicates the specific HDAC3 protein band, while the black ones indicate the specific domains of APPL1. C4‐2 cells were transfected with a pool of control or APPL1‐specific siRNAs for 48 h followed by IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cells were treated with 10 ng/ml of IGF‐1 for different periods of time followed by IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cells were transfected with indicated siRNAs and treated with 10 ng/ml of IGF‐1 for 30 min and followed by IP and Western blots with the indicated antibodies. The asterisks (*) indicate the specific HDAC3 protein bands. A hypothetical model depicting roles of HDAC3 and APPL1 in growth factor (GF)‐induced AKT activation. In the absence of the interaction of GF with a receptor tyrosine kinase (RTK), HDAC3 and APPL1 drift around in the cytosol. As a result, AKT becomes highly acetylated and resistant to be polyubiquitinated. Upon GF stimulation, RTK recruits APPL1, which in turn functions as a scaffold facilitating HDAC3‐mediated deacetylation of AKT, thereby making AKT poised for further activation by polyubiquitination. Activation of this deacetylase‐dependent function of HDAC3 may also require the binding by the deacetylase activating domain of SMRT. Source data are available online for this figure.
Figure Legend Snippet: The scaffold protein APPL 1 facilitates HDAC 3 regulation of AKT C4‐2 cell lysate was prepared for IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cell lysate was prepared for GST pull‐down assay using GST or GST‐HDAC3 recombinant proteins (stained with Coomassie blue, low panel) followed by Western blot with anti‐APPL1 antibody (upper panel). GST or GST‐HDAC3 recombinant proteins with expected molecular mass are indicated by asterisks. An illustration depicts four functional domains (BAR, PH, PTB, and PDZ) of APPL1 used for construction of GST‐APPL1 recombinant proteins. C4‐2 cell lysate was prepared for GST pull‐down assay using GST or GST‐APPL1 recombinant proteins (stained with Coomassie blue, low panel) followed by Western blot with anti‐HDAC3 antibody (upper panel). The red asterisk (*) indicates the specific HDAC3 protein band, while the black ones indicate the specific domains of APPL1. C4‐2 cells were transfected with a pool of control or APPL1‐specific siRNAs for 48 h followed by IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cells were treated with 10 ng/ml of IGF‐1 for different periods of time followed by IP and Western blots with the indicated antibodies. The asterisk (*) indicates the specific HDAC3 protein band. C4‐2 cells were transfected with indicated siRNAs and treated with 10 ng/ml of IGF‐1 for 30 min and followed by IP and Western blots with the indicated antibodies. The asterisks (*) indicate the specific HDAC3 protein bands. A hypothetical model depicting roles of HDAC3 and APPL1 in growth factor (GF)‐induced AKT activation. In the absence of the interaction of GF with a receptor tyrosine kinase (RTK), HDAC3 and APPL1 drift around in the cytosol. As a result, AKT becomes highly acetylated and resistant to be polyubiquitinated. Upon GF stimulation, RTK recruits APPL1, which in turn functions as a scaffold facilitating HDAC3‐mediated deacetylation of AKT, thereby making AKT poised for further activation by polyubiquitination. Activation of this deacetylase‐dependent function of HDAC3 may also require the binding by the deacetylase activating domain of SMRT. Source data are available online for this figure.

Techniques Used: Western Blot, Pull Down Assay, Recombinant, Staining, Functional Assay, Transfection, Activation Assay, Histone Deacetylase Assay, Binding Assay

HDAC 3 regulates AKT phosphorylation HDACIs inhibited AKT phosphorylation. C4‐2 cells were pre‐treated with 20 μM of CHX for 30 min followed by treatment with pan HDACIs TSA (1 μM), SAHA (5 μM), LBH589 (0.1 μM), or a HDAC6 selective inhibitor Tuba (5 μM) for 24 h prior to Western blot analysis with indicated antibodies. The efficacy of CHX was evident by blockade of induction of FBP1 expression by HDACIs as reported (Yang et al , 2017 ). The knockdown efficiency of each member of class I/II HDACs by shRNAs. C4‐2 cells were stably infected with control or gene‐specific shRNAs for 4 days and harvested for RT–qPCR. Expression of each gene was first normalized to the level of GAPDH , and then the expression level of each gene in gene knockdown cells was normalized by that in control knockdown cells. The shRNA knockdown efficiency was determined by subtracting the normalized value from 100%. Data represents means ± SEM. The RT–qPCR was performed in triplicate for each sample. Control or gene‐specific knockdown C4‐2 cells were serum starved for 24 h and then cultured in regular culture medium for 12 h followed by Western blots for indicated proteins. Western blot bands for total and phosphorylated AKT were quantified and normalized to the quantified values of β‐Tubulin (loading control). The normalized values were further normalized to the value of shC‐infected cells without serum stimulation. C4‐2 cells were treated with vehicle (DMSO) or HDAC3 inhibitor RGFP966, and at different time points, cells were harvested for Western blots with the indicated antibodies. The representatives of IHC staining for HDAC3 and AKT S473 phosphorylation in prostate cancer patient specimens; scale bar: 50 μm; scale bar for the inset: 20 μm. Correlation between expression of AKT S473 phosphorylation and HDAC3 was shown, n = 55, *** P = 1.87e‐07 was performed by Pearson's product‐moment correlation test. Source data are available online for this figure.
Figure Legend Snippet: HDAC 3 regulates AKT phosphorylation HDACIs inhibited AKT phosphorylation. C4‐2 cells were pre‐treated with 20 μM of CHX for 30 min followed by treatment with pan HDACIs TSA (1 μM), SAHA (5 μM), LBH589 (0.1 μM), or a HDAC6 selective inhibitor Tuba (5 μM) for 24 h prior to Western blot analysis with indicated antibodies. The efficacy of CHX was evident by blockade of induction of FBP1 expression by HDACIs as reported (Yang et al , 2017 ). The knockdown efficiency of each member of class I/II HDACs by shRNAs. C4‐2 cells were stably infected with control or gene‐specific shRNAs for 4 days and harvested for RT–qPCR. Expression of each gene was first normalized to the level of GAPDH , and then the expression level of each gene in gene knockdown cells was normalized by that in control knockdown cells. The shRNA knockdown efficiency was determined by subtracting the normalized value from 100%. Data represents means ± SEM. The RT–qPCR was performed in triplicate for each sample. Control or gene‐specific knockdown C4‐2 cells were serum starved for 24 h and then cultured in regular culture medium for 12 h followed by Western blots for indicated proteins. Western blot bands for total and phosphorylated AKT were quantified and normalized to the quantified values of β‐Tubulin (loading control). The normalized values were further normalized to the value of shC‐infected cells without serum stimulation. C4‐2 cells were treated with vehicle (DMSO) or HDAC3 inhibitor RGFP966, and at different time points, cells were harvested for Western blots with the indicated antibodies. The representatives of IHC staining for HDAC3 and AKT S473 phosphorylation in prostate cancer patient specimens; scale bar: 50 μm; scale bar for the inset: 20 μm. Correlation between expression of AKT S473 phosphorylation and HDAC3 was shown, n = 55, *** P = 1.87e‐07 was performed by Pearson's product‐moment correlation test. Source data are available online for this figure.

Techniques Used: Western Blot, Expressing, Stable Transfection, Infection, Quantitative RT-PCR, shRNA, Cell Culture, Immunohistochemistry, Staining

20) Product Images from "Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function"

Article Title: Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01615

Histone deacetylases (HDAC) inhibitors regulate cytokine production. Expressions of IFN-γ and TNF-α were detected by quantitative real-time PCR and sandwich ELISA. (A,B) IFN-γ expression by γδ T cells stimulated with HDMAPP, treated with or without HDAC inhibitors sodium valproate (VPA), Trichostatin-A (TSA), and suberoylanilidehydroxamic acid (SAHA) at different concentrations at mRNA and protein levels, respectively. (C,D) Expression of TNF-α in the supernatants collected from HDMAPP stimulated γδ T cells in the presence or absence of HDAC inhibitors VPA, TSA, and SAHA at different concentrations at mRNA and protein levels, respectively. The expression of different m-RNA transcripts was normalized to 18S r-RNA. All the results indicated are mean ± SEM of three independent experiments, where * p
Figure Legend Snippet: Histone deacetylases (HDAC) inhibitors regulate cytokine production. Expressions of IFN-γ and TNF-α were detected by quantitative real-time PCR and sandwich ELISA. (A,B) IFN-γ expression by γδ T cells stimulated with HDMAPP, treated with or without HDAC inhibitors sodium valproate (VPA), Trichostatin-A (TSA), and suberoylanilidehydroxamic acid (SAHA) at different concentrations at mRNA and protein levels, respectively. (C,D) Expression of TNF-α in the supernatants collected from HDMAPP stimulated γδ T cells in the presence or absence of HDAC inhibitors VPA, TSA, and SAHA at different concentrations at mRNA and protein levels, respectively. The expression of different m-RNA transcripts was normalized to 18S r-RNA. All the results indicated are mean ± SEM of three independent experiments, where * p

Techniques Used: Real-time Polymerase Chain Reaction, Sandwich ELISA, Expressing

Histone deacetylases (HDAC) inhibitors increase the expression of cell cycle checkpoint proteins p53 and p21. Protein expression of p53 and p21 by γδ T cells upon treatment with (A) sodium valproate (VPA), (B) Trichostatin-A (TSA), and (C) suberoylanilidehydroxamic acid (SAHA) as detected by western blotting. Cell lysates of γδ T cells, stimulated with HDMAPP after treatment with HDAC inhibitors at different concentrations for 72 h were prepared and p53, p21 proteins were detected. β-actin was used as loading control. Densitometry quantification of p53 (D–F) and p21 (G–I) expression in γδ T cells upon treatment with VPA, TSA, and SAHA, relative to β-actin.
Figure Legend Snippet: Histone deacetylases (HDAC) inhibitors increase the expression of cell cycle checkpoint proteins p53 and p21. Protein expression of p53 and p21 by γδ T cells upon treatment with (A) sodium valproate (VPA), (B) Trichostatin-A (TSA), and (C) suberoylanilidehydroxamic acid (SAHA) as detected by western blotting. Cell lysates of γδ T cells, stimulated with HDMAPP after treatment with HDAC inhibitors at different concentrations for 72 h were prepared and p53, p21 proteins were detected. β-actin was used as loading control. Densitometry quantification of p53 (D–F) and p21 (G–I) expression in γδ T cells upon treatment with VPA, TSA, and SAHA, relative to β-actin.

Techniques Used: Expressing, Western Blot

21) Product Images from "Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats"

Article Title: Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113539

Histone deacetylase (HDAC) inhibitors reduce the expression of G6P and PCK1 in HepG2 cells. Cells were treated with VPA (( A , F ), 0.1, 1.0, and 10 mmol/L); SAHA (( B , G ), 0.1, 1.0, and 10 µmol/L); TSA ( C , H , 0.1, 0.3, and 1.0 µmol/L); MS275 (( D , I ), 1.0, 10, and 100 µmol/L); and MC1568 (( E , J ), 0.1, 1.0, and 10 µmol/L) for 48 h under hyperglycemic conditions. Expression of G6P or PCK1 was quantified by RT-qPCR. Cultivation under hyperglycemic conditions increased the expression of G6P or PCK1 , an effect that was attenuated by treatment with the pan-HDAC inhibitors VPA, SAHA, and TSA, as well as the HDAC class I-specific inhibitor, MS275, and the HDAC class IIa-specific inhibitor, MC1568. The graphs show the mean ± SEM of three independent experiments (* p
Figure Legend Snippet: Histone deacetylase (HDAC) inhibitors reduce the expression of G6P and PCK1 in HepG2 cells. Cells were treated with VPA (( A , F ), 0.1, 1.0, and 10 mmol/L); SAHA (( B , G ), 0.1, 1.0, and 10 µmol/L); TSA ( C , H , 0.1, 0.3, and 1.0 µmol/L); MS275 (( D , I ), 1.0, 10, and 100 µmol/L); and MC1568 (( E , J ), 0.1, 1.0, and 10 µmol/L) for 48 h under hyperglycemic conditions. Expression of G6P or PCK1 was quantified by RT-qPCR. Cultivation under hyperglycemic conditions increased the expression of G6P or PCK1 , an effect that was attenuated by treatment with the pan-HDAC inhibitors VPA, SAHA, and TSA, as well as the HDAC class I-specific inhibitor, MS275, and the HDAC class IIa-specific inhibitor, MC1568. The graphs show the mean ± SEM of three independent experiments (* p

Techniques Used: Histone Deacetylase Assay, Expressing, Quantitative RT-PCR

22) Product Images from "Molecular Characterization of Propionyllysines in Non-histone Proteins *Molecular Characterization of Propionyllysines in Non-histone Proteins * S⃞"

Article Title: Molecular Characterization of Propionyllysines in Non-histone Proteins *Molecular Characterization of Propionyllysines in Non-histone Proteins * S⃞

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M800224-MCP200

p300 and CBP are lysine propionylated in vivo . A , the p300 propionylation was induced in vivo by HDAC inhibitor mixture. 293T cells were transfected to express FLAG-p300. The indicated transfection cells were treated with HDAC inhibitor mixture for six
Figure Legend Snippet: p300 and CBP are lysine propionylated in vivo . A , the p300 propionylation was induced in vivo by HDAC inhibitor mixture. 293T cells were transfected to express FLAG-p300. The indicated transfection cells were treated with HDAC inhibitor mixture for six

Techniques Used: In Vivo, Transfection

23) Product Images from "Ability of plasma-activated acetated Ringer’s solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors"

Article Title: Ability of plasma-activated acetated Ringer’s solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.19-104

Effects of HDAC inhibitors on PAA-triggered mitochondrial injury. (A) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, followed by an assay for ROS accumulation. Data are shown as means ± SD ( n = 3). ** p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-triggered mitochondrial injury. (A) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, followed by an assay for ROS accumulation. Data are shown as means ± SD ( n = 3). ** p

Techniques Used:

Effects of HDAC inhibitors on PAA-triggered nuclear injury. (A) A549 cells were treated with or without TSA (1 µM) or VPA (2 mM) for 12 h and their acetylated histones were detected by Western blotting. Protein concentrations in samples were confirmed by Coomassie brilliant blue (CBB) staining. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in a CO 2 incubator, followed by Western blotting for γH2AX. (C) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in the presence or absence of DPQ (10 µM), followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-triggered nuclear injury. (A) A549 cells were treated with or without TSA (1 µM) or VPA (2 mM) for 12 h and their acetylated histones were detected by Western blotting. Protein concentrations in samples were confirmed by Coomassie brilliant blue (CBB) staining. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in a CO 2 incubator, followed by Western blotting for γH2AX. (C) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in the presence or absence of DPQ (10 µM), followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p

Techniques Used: Western Blot, Staining, MTT Assay

Effects of HDAC inhibitors on PAA-induced cell injury. (A) A scheme depicting the experimental set-up and preparation of PAA. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) in DMEM-10% FCS for 12 h and then with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, and this was followed by an assay for PI-stained cells by flow cytometry as described in the Materials and methods section. PI-positive cell counts were analyzed (right). Data are shown as means ± SD ( n = 3). (C) A549 cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 6 or 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-induced cell injury. (A) A scheme depicting the experimental set-up and preparation of PAA. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) in DMEM-10% FCS for 12 h and then with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, and this was followed by an assay for PI-stained cells by flow cytometry as described in the Materials and methods section. PI-positive cell counts were analyzed (right). Data are shown as means ± SD ( n = 3). (C) A549 cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 6 or 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p

Techniques Used: Staining, Flow Cytometry, MTT Assay

Effects of HDAC inhibitors and PAA on the viability of normal cells. HaCaT cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data for A549 cells are quoted from Fig. 1 C right. Data are shown as means ± SD ( n = 3). ** p
Figure Legend Snippet: Effects of HDAC inhibitors and PAA on the viability of normal cells. HaCaT cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data for A549 cells are quoted from Fig. 1 C right. Data are shown as means ± SD ( n = 3). ** p

Techniques Used: MTT Assay

24) Product Images from "Ability of plasma-activated acetated Ringer’s solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors"

Article Title: Ability of plasma-activated acetated Ringer’s solution to induce A549 cell injury is enhanced by a pre-treatment with histone deacetylase inhibitors

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.19-104

Effects of HDAC inhibitors on PAA-triggered mitochondrial injury. (A) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, followed by an assay for ROS accumulation. Data are shown as means ± SD ( n = 3). ** p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-triggered mitochondrial injury. (A) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, followed by an assay for ROS accumulation. Data are shown as means ± SD ( n = 3). ** p

Techniques Used:

Effects of HDAC inhibitors on PAA-triggered nuclear injury. (A) A549 cells were treated with or without TSA (1 µM) or VPA (2 mM) for 12 h and their acetylated histones were detected by Western blotting. Protein concentrations in samples were confirmed by Coomassie brilliant blue (CBB) staining. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in a CO 2 incubator, followed by Western blotting for γH2AX. (C) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in the presence or absence of DPQ (10 µM), followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-triggered nuclear injury. (A) A549 cells were treated with or without TSA (1 µM) or VPA (2 mM) for 12 h and their acetylated histones were detected by Western blotting. Protein concentrations in samples were confirmed by Coomassie brilliant blue (CBB) staining. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in a CO 2 incubator, followed by Western blotting for γH2AX. (C) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) for 12 h and were then treated with Ac-R (v) or PAA for 2 h in the presence or absence of DPQ (10 µM), followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p

Techniques Used: Western Blot, Staining, MTT Assay

Effects of HDAC inhibitors on PAA-induced cell injury. (A) A scheme depicting the experimental set-up and preparation of PAA. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) in DMEM-10% FCS for 12 h and then with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, and this was followed by an assay for PI-stained cells by flow cytometry as described in the Materials and methods section. PI-positive cell counts were analyzed (right). Data are shown as means ± SD ( n = 3). (C) A549 cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 6 or 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p
Figure Legend Snippet: Effects of HDAC inhibitors on PAA-induced cell injury. (A) A scheme depicting the experimental set-up and preparation of PAA. (B) A549 cells were pretreated with or without TSA (1 µM) or VPA (2 mM) in DMEM-10% FCS for 12 h and then with Ac-R (vehicle) or PAA for 1 h in a CO 2 incubator, and this was followed by an assay for PI-stained cells by flow cytometry as described in the Materials and methods section. PI-positive cell counts were analyzed (right). Data are shown as means ± SD ( n = 3). (C) A549 cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 6 or 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data are shown as means ± SD ( n = 3). * p

Techniques Used: Staining, Flow Cytometry, MTT Assay

Effects of HDAC inhibitors and PAA on the viability of normal cells. HaCaT cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data for A549 cells are quoted from Fig. 1 C right. Data are shown as means ± SD ( n = 3). ** p
Figure Legend Snippet: Effects of HDAC inhibitors and PAA on the viability of normal cells. HaCaT cells were pretreated with or without the indicated concentrations of TSA or VPA in DMEM-10% FCS for 12 h and were then treated with Ac-R (vehicle) or PAA for 2 h in a CO 2 incubator, followed by an assay for cell viability by the MTT assay. Data for A549 cells are quoted from Fig. 1 C right. Data are shown as means ± SD ( n = 3). ** p

Techniques Used: MTT Assay

25) Product Images from "Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells"

Article Title: Butyrate produced by gut commensal bacteria activates TGF-beta1 expression through the transcription factor SP1 in human intestinal epithelial cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-28048-y

HDAC inhibitor mimicked the butyrate-dependent activation of TGFB1 expression. HT-29-TGF prom reporter cells were incubated with butyrate (2 mM), SAHA (5 μM), trichostatin A, (TSA, 1 μM) or controls (DMSO and RPMI). TGFB1 expression was measured by luciferase activity and expressed as fold increase towards the control: un-stimulated cells. Data are represented as median ± quartiles of fold change towards un-stimulated cells. N ≥ 3, Wilcoxon test, *P
Figure Legend Snippet: HDAC inhibitor mimicked the butyrate-dependent activation of TGFB1 expression. HT-29-TGF prom reporter cells were incubated with butyrate (2 mM), SAHA (5 μM), trichostatin A, (TSA, 1 μM) or controls (DMSO and RPMI). TGFB1 expression was measured by luciferase activity and expressed as fold increase towards the control: un-stimulated cells. Data are represented as median ± quartiles of fold change towards un-stimulated cells. N ≥ 3, Wilcoxon test, *P

Techniques Used: Activation Assay, Expressing, Incubation, Luciferase, Activity Assay

26) Product Images from "Determinants of the Establishment of Human Immunodeficiency Virus Type 1 Latency ▿"

Article Title: Determinants of the Establishment of Human Immunodeficiency Virus Type 1 Latency ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02058-08

HDAC inhibitors do not prevent latency establishment. Jurkat cells were pretreated for 16 h with NaBu (100 μM) and infected with an HIV-1 EGFP reporter virus. Levels of silent infection on day 3 p.i. and levels of latent infection on day 37 were
Figure Legend Snippet: HDAC inhibitors do not prevent latency establishment. Jurkat cells were pretreated for 16 h with NaBu (100 μM) and infected with an HIV-1 EGFP reporter virus. Levels of silent infection on day 3 p.i. and levels of latent infection on day 37 were

Techniques Used: Infection

27) Product Images from "Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells"

Article Title: Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-37019-2

Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P
Figure Legend Snippet: Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P

Techniques Used: Expressing, Activation Assay, Luciferase, Activity Assay

28) Product Images from "Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells"

Article Title: Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-37019-2

Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P
Figure Legend Snippet: Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P

Techniques Used: Expressing, Activation Assay, Luciferase, Activity Assay

29) Product Images from "Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function"

Article Title: Checkpoint Blockade Rescues the Repressive Effect of Histone Deacetylases Inhibitors on γδ T Cell Function

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01615

Histone deacetylases (HDAC) inhibitors regulate cytokine production. Expressions of IFN-γ and TNF-α were detected by quantitative real-time PCR and sandwich ELISA. (A,B) IFN-γ expression by γδ T cells stimulated with HDMAPP, treated with or without HDAC inhibitors sodium valproate (VPA), Trichostatin-A (TSA), and suberoylanilidehydroxamic acid (SAHA) at different concentrations at mRNA and protein levels, respectively. (C,D) Expression of TNF-α in the supernatants collected from HDMAPP stimulated γδ T cells in the presence or absence of HDAC inhibitors VPA, TSA, and SAHA at different concentrations at mRNA and protein levels, respectively. The expression of different m-RNA transcripts was normalized to 18S r-RNA. All the results indicated are mean ± SEM of three independent experiments, where * p
Figure Legend Snippet: Histone deacetylases (HDAC) inhibitors regulate cytokine production. Expressions of IFN-γ and TNF-α were detected by quantitative real-time PCR and sandwich ELISA. (A,B) IFN-γ expression by γδ T cells stimulated with HDMAPP, treated with or without HDAC inhibitors sodium valproate (VPA), Trichostatin-A (TSA), and suberoylanilidehydroxamic acid (SAHA) at different concentrations at mRNA and protein levels, respectively. (C,D) Expression of TNF-α in the supernatants collected from HDMAPP stimulated γδ T cells in the presence or absence of HDAC inhibitors VPA, TSA, and SAHA at different concentrations at mRNA and protein levels, respectively. The expression of different m-RNA transcripts was normalized to 18S r-RNA. All the results indicated are mean ± SEM of three independent experiments, where * p

Techniques Used: Real-time Polymerase Chain Reaction, Sandwich ELISA, Expressing

Histone deacetylases (HDAC) inhibitors increase the expression of cell cycle checkpoint proteins p53 and p21. Protein expression of p53 and p21 by γδ T cells upon treatment with (A) sodium valproate (VPA), (B) Trichostatin-A (TSA), and (C) suberoylanilidehydroxamic acid (SAHA) as detected by western blotting. Cell lysates of γδ T cells, stimulated with HDMAPP after treatment with HDAC inhibitors at different concentrations for 72 h were prepared and p53, p21 proteins were detected. β-actin was used as loading control. Densitometry quantification of p53 (D–F) and p21 (G–I) expression in γδ T cells upon treatment with VPA, TSA, and SAHA, relative to β-actin.
Figure Legend Snippet: Histone deacetylases (HDAC) inhibitors increase the expression of cell cycle checkpoint proteins p53 and p21. Protein expression of p53 and p21 by γδ T cells upon treatment with (A) sodium valproate (VPA), (B) Trichostatin-A (TSA), and (C) suberoylanilidehydroxamic acid (SAHA) as detected by western blotting. Cell lysates of γδ T cells, stimulated with HDMAPP after treatment with HDAC inhibitors at different concentrations for 72 h were prepared and p53, p21 proteins were detected. β-actin was used as loading control. Densitometry quantification of p53 (D–F) and p21 (G–I) expression in γδ T cells upon treatment with VPA, TSA, and SAHA, relative to β-actin.

Techniques Used: Expressing, Western Blot

30) Product Images from "Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes"

Article Title: Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes

Journal: Haematologica

doi: 10.3324/haematol.2008.001933

The effect on leukemic cells of the PARP inhibitor, KU, in combination with non-cytotoxic concentrations of 5’-aza-2’-dCR and/or HDAC inhibitor. (A) The effect of the PARP inhibitor, KU, in combination with the HDAC inhibitor, MS275, and
Figure Legend Snippet: The effect on leukemic cells of the PARP inhibitor, KU, in combination with non-cytotoxic concentrations of 5’-aza-2’-dCR and/or HDAC inhibitor. (A) The effect of the PARP inhibitor, KU, in combination with the HDAC inhibitor, MS275, and

Techniques Used:

The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top
Figure Legend Snippet: The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top

Techniques Used: Immunostaining, Staining

31) Product Images from "Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence"

Article Title: Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence

Journal: Genes & Development

doi: 10.1101/gad.331272.119

HDAC inhibitors improve mitochondria function and suppress formation of CCFs and expression of the SASP. ( A ) Scheme of experimental design in B – M . ( B ) Proliferating or senescent (IR) IMR90 cells treated with TSA at indicated dose range (20–50–100 nM) were analyzed for H4K16ac and H4 by immunoblotting. ( C ) The effect of TSA (100 nM) on cell proliferation was assessed by EdU incorporation. Representative fluorescent images and quantification are shown. Scale bar, 10 μm. ( D ) Lysates from proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. ( E ) Relative fold change in nuclear-encoded oxidative phosphorylation genes (KEGG hsa00190; subunits of complexes I–V) was represented in a histogram with bin size of 0.1 log 2 fold change units. Counts in each bin were normalized to total number of nuclear-encoded mitochondrial genes detected. ( F ) The expression of the indicated nuclear-encoded mitochondrial genes was analyzed by RT-qPCR in senescent cells (IR) treated with or without TSA (100 nM). ( G ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. ( H ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA were incubated with TMRE (100 nM) and Mitotracker Green (MTG; 100 nM) and analyzed by flow cytometry. The ratio of TMRE and MTG fluorescence intensity was used to represent mitochondrial membrane potential (MMP). ( I . ( J ) Expression of ROS-responsive gene SOD2 was determined by RT-qPCR in proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM). ( K , L ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were imaged by confocal microscope and quantified for formation of γH2AX-positive CCFs (indicated by arrows) ( K ) or monitored for the expression of indicated SASP genes by RT-qPCR ( L ). Scale bar, 8 μm. Data shown in C , F , and H – L are mean ± SEM of three independent experiments. Statistical analysis was performed with unpaired two-tailed Student's t -test. (****) P
Figure Legend Snippet: HDAC inhibitors improve mitochondria function and suppress formation of CCFs and expression of the SASP. ( A ) Scheme of experimental design in B – M . ( B ) Proliferating or senescent (IR) IMR90 cells treated with TSA at indicated dose range (20–50–100 nM) were analyzed for H4K16ac and H4 by immunoblotting. ( C ) The effect of TSA (100 nM) on cell proliferation was assessed by EdU incorporation. Representative fluorescent images and quantification are shown. Scale bar, 10 μm. ( D ) Lysates from proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. ( E ) Relative fold change in nuclear-encoded oxidative phosphorylation genes (KEGG hsa00190; subunits of complexes I–V) was represented in a histogram with bin size of 0.1 log 2 fold change units. Counts in each bin were normalized to total number of nuclear-encoded mitochondrial genes detected. ( F ) The expression of the indicated nuclear-encoded mitochondrial genes was analyzed by RT-qPCR in senescent cells (IR) treated with or without TSA (100 nM). ( G ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were analyzed for the indicated proteins by immunoblotting. ( H ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA were incubated with TMRE (100 nM) and Mitotracker Green (MTG; 100 nM) and analyzed by flow cytometry. The ratio of TMRE and MTG fluorescence intensity was used to represent mitochondrial membrane potential (MMP). ( I . ( J ) Expression of ROS-responsive gene SOD2 was determined by RT-qPCR in proliferating (Prolif) and senescent (IR) IMR90 cells with or without TSA (100 nM). ( K , L ) Proliferating (Prolif) and senescent (IR) cells treated with or without TSA (100 nM) were imaged by confocal microscope and quantified for formation of γH2AX-positive CCFs (indicated by arrows) ( K ) or monitored for the expression of indicated SASP genes by RT-qPCR ( L ). Scale bar, 8 μm. Data shown in C , F , and H – L are mean ± SEM of three independent experiments. Statistical analysis was performed with unpaired two-tailed Student's t -test. (****) P

Techniques Used: Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry, Fluorescence, Microscopy, Two Tailed Test

32) Product Images from "Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells"

Article Title: Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02838

HDAC inhibitor mimicked the butyrate-dependent down-regulation of IDO-1 expression in a SP1, PPARγ and AP-1 independent manner. (A) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2 mM), SAHA (5 μM), Trichostatin A (TSA 1 μM) or Valproic acid (VAP 5 mM) ± SP1 inhibitor (Mitramycin A; MitA 0.1 μM). (B) , HT-29- IDO-1 reporter cells were stimulated for 24 h with two PPARγ activators: Pioglitazone (Pio 5 μM); Rosiglitazone (Rosi, 10 μM) or the specific PPARγ inhibitor GW9662 (10 μM) ± butyrate (But 2 mM). (C) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2mM) and/or the AP1 inhibitor, SR11302 (10 μM). IDO-1 expression was measured by luciferase activity and expressed as median ± quartiles of fold change toward un-stimulated cells. Data represented at least three independent experiments. P -value: *** P
Figure Legend Snippet: HDAC inhibitor mimicked the butyrate-dependent down-regulation of IDO-1 expression in a SP1, PPARγ and AP-1 independent manner. (A) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2 mM), SAHA (5 μM), Trichostatin A (TSA 1 μM) or Valproic acid (VAP 5 mM) ± SP1 inhibitor (Mitramycin A; MitA 0.1 μM). (B) , HT-29- IDO-1 reporter cells were stimulated for 24 h with two PPARγ activators: Pioglitazone (Pio 5 μM); Rosiglitazone (Rosi, 10 μM) or the specific PPARγ inhibitor GW9662 (10 μM) ± butyrate (But 2 mM). (C) HT-29- IDO-1 reporter cells were incubated for 24 h with butyrate (But 2mM) and/or the AP1 inhibitor, SR11302 (10 μM). IDO-1 expression was measured by luciferase activity and expressed as median ± quartiles of fold change toward un-stimulated cells. Data represented at least three independent experiments. P -value: *** P

Techniques Used: Expressing, Incubation, Luciferase, Activity Assay

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    Millipore trichostatin a
    Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. <t>Trichostatin</t> A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P
    Trichostatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trichostatin a/product/Millipore
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    Millipore hdac inhibitors
    The effect of PARP and <t>HDAC</t> inhibitors on γH2AX-P foci, as indicators of double-strand <t>DNA</t> break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top
    Hdac Inhibitors, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P

    Journal: Scientific Reports

    Article Title: Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells

    doi: 10.1038/s41598-018-37019-2

    Figure Lengend Snippet: Impact of HDAC inhibitors on AhR reporter system and CYP1A1 . HT29 ( A ) and Caco-2 ( B ) -AhR reporter cells were stimulated with HDAC inhibitors for 24 h. Trichostatin A (TSA, 0.1 μM and 1 μM), Vorinostat (SAHA 5 µM), Valproic acid (VAP 5 mM), butyrate (But, 2 mM) and TCDD (10 nM). ( C ) Relative expression to control of CYP1A1 on HT-29 cells treated with butyrate 2 mM, TCDD 10 nM or TSA 0.1 μM during 6 h. CYP1A1 expression induced by drugs is expressed as relative to control expression and is determined by the 2 −ΔΔCt method using β-ACTIN as control gene. AhR activation was measured by luciferase activity and expressed as fold increase means (±SEM) of at least three independent experiments, normalised on un-treated cells. ns: P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P

    Article Snippet: HDAC inhibitors: Trichostatin A (TSA 0.1 and 1 µM, Sigma), vorinostat (SAHA 5 µM, Sigma) and valproic acid (VPA 5 mM, Sigma).

    Techniques: Expressing, Activation Assay, Luciferase, Activity Assay

    miR-137 is hypermethylated and reactivated by epigenetic inhibitors in endometrial cancer cells. ( a ) DNA methylation levels determined by bisulfite pyrosequencing in a normal endometrial cell line (EM-E6/E7/TERT) and 11 endometrial cancer cell lines. ( b–c ) Reactivation of pre-/pri- and mature miR-137 after epigenetic inhibitors in endometrial cancer cells after endometrial cancer cells were treated with epigenetic inhibitors. Cancer cells (HEC1A, Ishikawa H, and Hec50co) were treated with 5-aza-2′-deoxycytidine (DAC) and/or trichostatin A (TSA). Gene expression was determined by RT-qPCR and compared to untreated controls (CTR). U6 served as an internal control. Bars: means±SD; *: P

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

    doi: 10.1038/s41374-018-0092-x

    Figure Lengend Snippet: miR-137 is hypermethylated and reactivated by epigenetic inhibitors in endometrial cancer cells. ( a ) DNA methylation levels determined by bisulfite pyrosequencing in a normal endometrial cell line (EM-E6/E7/TERT) and 11 endometrial cancer cell lines. ( b–c ) Reactivation of pre-/pri- and mature miR-137 after epigenetic inhibitors in endometrial cancer cells after endometrial cancer cells were treated with epigenetic inhibitors. Cancer cells (HEC1A, Ishikawa H, and Hec50co) were treated with 5-aza-2′-deoxycytidine (DAC) and/or trichostatin A (TSA). Gene expression was determined by RT-qPCR and compared to untreated controls (CTR). U6 served as an internal control. Bars: means±SD; *: P

    Article Snippet: To assess the role of DNA methylation in miR-137 expression, endometrial cancer cells were treated with 5-aza-2′-deoxycytidine (DAC; 5 μmol/L) for 48 hours, with or without trichostatin A (TSA, 0.5 μmol/L) (Sigma-Aldrich, St. Louis MO) for 24 hours.

    Techniques: DNA Methylation Assay, Expressing, Quantitative RT-PCR

    The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top

    Journal: Haematologica

    Article Title: Inhibitors of poly ADP-ribose polymerase (PARP) induce apoptosis of myeloid leukemic cells: potential for therapy of myeloid leukemia and myelodysplastic syndromes

    doi: 10.3324/haematol.2008.001933

    Figure Lengend Snippet: The effect of PARP and HDAC inhibitors on γH2AX-P foci, as indicators of double-strand DNA break repair. (A) Immunostaining of nuclei (DAPI stained in blue) from P39 cells treated for 24 h with 1 μM KU (top left panel); 100 nM MS275 (top

    Article Snippet: HDAC inhibitors, trichostatin A, sodium butyrate, and the DNA methyltransferase inhibitor, 5’ aza 2’deoxycytidine (5-aza-2’CdR), were purchased from Sigma Biochemicals, Poole, UK.

    Techniques: Immunostaining, Staining

    HDAC inhibition stimulates SUMOylation in cardiac cells. (A) Neonatal rat ventricular myocytes (NRVMs) were treated with the pan-HDAC inhibitor trichostatin A (TSA) for the indicated amounts of time. SUMO-1 conjugates were examined by immunoblotting.

    Journal: Cellular signalling

    Article Title: Class I HDAC Inhibition Stimulates Cardiac Protein SUMOylation Through a Post-Translational Mechanism

    doi: 10.1016/j.cellsig.2014.09.005

    Figure Lengend Snippet: HDAC inhibition stimulates SUMOylation in cardiac cells. (A) Neonatal rat ventricular myocytes (NRVMs) were treated with the pan-HDAC inhibitor trichostatin A (TSA) for the indicated amounts of time. SUMO-1 conjugates were examined by immunoblotting.

    Article Snippet: HDAC inhibitors: trichostatin A (TSA) (Sigma; 100 nM), MGCD0103 (Selleck; 1 μM), diphenylacetohydroxamic acid (DPAH) (Sigma [D6071]; 10 μM), tubastatin A (Selleck; 1μM), MS-275 (Selleck; 1μM).

    Techniques: Inhibition