hdac inhibitors  (Selleck Chemicals)


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    Selleck Chemicals hdac inhibitors
    Neither <t>HDAC</t> nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM <t>vorinostat</t> for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).
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    1) Product Images from "Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors"

    Article Title: Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

    Journal: Oncotarget

    doi:

    Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).
    Figure Legend Snippet: Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).

    Techniques Used: Activity Assay, Transfection, Expressing, Glo Assay

    BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).
    Figure Legend Snippet: BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).

    Techniques Used: Glo Assay, Transfection, Expressing

    2) Product Images from "CG200745, an HDAC inhibitor, induces anti-tumour effects in cholangiocarcinoma cell lines via miRNAs targeting the Hippo pathway"

    Article Title: CG200745, an HDAC inhibitor, induces anti-tumour effects in cholangiocarcinoma cell lines via miRNAs targeting the Hippo pathway

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11094-3

    Anti-proliferative activities of HDAC inhibitors against SNU-1196, SNU-1196/GR, and SNU-308 cells. Cell viability was assessed with the MTT assay. ( a ) IC 50 values of CG200745 were 0.6, 0.9, and 1.8 μM, respectively; ( b ) IC 50 values of entinostat were 16.4, 48.8, and 6.7 μM, respectively; and ( c ) IC 50 values of vorinostat were 1.2, 2.6, and 3.9 μM, respectively. In SNU-1196 and SNU-1196/GR cells, ( d ) CG200745 (IC 50 ) induced the expression of apoptosis-related proteins p21 and BAX, as determined by western blotting; ( e ) CG200745 (IC 50 ) inhibited the expression of multidrug resistance genes, as determined by PCR; and ( f ) CG200745 (IC 50 ) altered the expression level of HDAC isozyme, as determined by western blotting.
    Figure Legend Snippet: Anti-proliferative activities of HDAC inhibitors against SNU-1196, SNU-1196/GR, and SNU-308 cells. Cell viability was assessed with the MTT assay. ( a ) IC 50 values of CG200745 were 0.6, 0.9, and 1.8 μM, respectively; ( b ) IC 50 values of entinostat were 16.4, 48.8, and 6.7 μM, respectively; and ( c ) IC 50 values of vorinostat were 1.2, 2.6, and 3.9 μM, respectively. In SNU-1196 and SNU-1196/GR cells, ( d ) CG200745 (IC 50 ) induced the expression of apoptosis-related proteins p21 and BAX, as determined by western blotting; ( e ) CG200745 (IC 50 ) inhibited the expression of multidrug resistance genes, as determined by PCR; and ( f ) CG200745 (IC 50 ) altered the expression level of HDAC isozyme, as determined by western blotting.

    Techniques Used: MTT Assay, Expressing, Western Blot, Polymerase Chain Reaction

    3) Product Images from "PU.1 Suppresses Th2 Cytokine Expression via Silencing of GATA3 Transcription in Dendritic Cells"

    Article Title: PU.1 Suppresses Th2 Cytokine Expression via Silencing of GATA3 Transcription in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137699

    Effects of PU.1 knockdown on the histone acetylation and on HDAC recruitment at the Gata3-1b promoter. Histone acetylation status of the Gata3-1b promoter in BMDCs transfected with either PU.1 siRNA (siPU.1) or its control siRNA (siCTRL) (A), and the acetylation status in BMDCs cultured without (CTRL) or with 10 nM the histone deacetylation inhibitor trichostatin A (TSA) for 3 h (B). Quantification of acetyl-histone H3 at the Gata3-1b promoter was performed by ChIP assay. (C, D) GATA3 mRNA levels in BMDCs cultured with 10 nM trichostatin A for the indicated times (C) or with the indicated trichostatin A concentration for 3 h (D). Effects of HDAC inhibitors (E) and knockdown of HDACs (F) on GATA3 mRNA levels. BMDCs were treated with MS-275 (1 μM, 10 μM), Droxinostat (20 μM, 50 μM), or MC1568 (5 μM, 20 μM) for 6 h (E). Relative mRNA levels (GATA3-1b/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. (G) The binding degree of HDAC3 on the Gata3-1b promoter was analyzed by a ChIP assay. Open circles, control rabbit IgG binding in control siRNA-transfected cells; closed circles, anti-HDAC3 antibody binding in control cells; open squares, control IgG binding in PU.1 siRNA-transfected cells; closed squares, anti-HDAC3 antibody binding in PU.1 siRNA-transfected cells. All results are means ± S.E.s ( n = 3). Similar results were obtained in three separate experiments. *, p
    Figure Legend Snippet: Effects of PU.1 knockdown on the histone acetylation and on HDAC recruitment at the Gata3-1b promoter. Histone acetylation status of the Gata3-1b promoter in BMDCs transfected with either PU.1 siRNA (siPU.1) or its control siRNA (siCTRL) (A), and the acetylation status in BMDCs cultured without (CTRL) or with 10 nM the histone deacetylation inhibitor trichostatin A (TSA) for 3 h (B). Quantification of acetyl-histone H3 at the Gata3-1b promoter was performed by ChIP assay. (C, D) GATA3 mRNA levels in BMDCs cultured with 10 nM trichostatin A for the indicated times (C) or with the indicated trichostatin A concentration for 3 h (D). Effects of HDAC inhibitors (E) and knockdown of HDACs (F) on GATA3 mRNA levels. BMDCs were treated with MS-275 (1 μM, 10 μM), Droxinostat (20 μM, 50 μM), or MC1568 (5 μM, 20 μM) for 6 h (E). Relative mRNA levels (GATA3-1b/GAPDH) were determined by quantitative RT-PCR after normalizing to GAPDH mRNA. (G) The binding degree of HDAC3 on the Gata3-1b promoter was analyzed by a ChIP assay. Open circles, control rabbit IgG binding in control siRNA-transfected cells; closed circles, anti-HDAC3 antibody binding in control cells; open squares, control IgG binding in PU.1 siRNA-transfected cells; closed squares, anti-HDAC3 antibody binding in PU.1 siRNA-transfected cells. All results are means ± S.E.s ( n = 3). Similar results were obtained in three separate experiments. *, p

    Techniques Used: Transfection, Cell Culture, Chromatin Immunoprecipitation, Concentration Assay, Mass Spectrometry, Quantitative RT-PCR, Binding Assay

    4) Product Images from "Class I and II Histone Deacetylase Inhibitors Differentially Regulate Thermogenic Gene Expression in Brown Adipocytes"

    Article Title: Class I and II Histone Deacetylase Inhibitors Differentially Regulate Thermogenic Gene Expression in Brown Adipocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31560-w

    Class II HDAC inhibitor MC-1568 inhibits the expression of Ucp1 , Pparγ and Prdm16 , but stimulates the expression of Pgc1α , Pgc1β , Acox1 and Cidea in brown adipocytes. HIB-1B cells were pre-treated with MC-1568 (10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Gene expression was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; Bars with different letters are significantly different from each other in control groups (without NE) or NE-treated groups.
    Figure Legend Snippet: Class II HDAC inhibitor MC-1568 inhibits the expression of Ucp1 , Pparγ and Prdm16 , but stimulates the expression of Pgc1α , Pgc1β , Acox1 and Cidea in brown adipocytes. HIB-1B cells were pre-treated with MC-1568 (10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Gene expression was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; Bars with different letters are significantly different from each other in control groups (without NE) or NE-treated groups.

    Techniques Used: Expressing, Quantitative RT-PCR

    Pan-HDAC inhibitor TSA exhibits a mixed effect on the expression of brown fat genes. TSA inhibits the expression of brown fat genes Ucp1 ( A ), Pparγ ( B ), and Prdm16 ( C ), while increasing others including Pgc1α ( D ), Pgc1β ( E ), Acox1 ( F ), and Cidea ( G ) in HIB-1B cells. TSA has no effects on Cox1 ( H ) and Cpt1b ( I ) expression. Cells were pre-treated with TSA (500 nM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Brown adipocyte mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 4–6; *p
    Figure Legend Snippet: Pan-HDAC inhibitor TSA exhibits a mixed effect on the expression of brown fat genes. TSA inhibits the expression of brown fat genes Ucp1 ( A ), Pparγ ( B ), and Prdm16 ( C ), while increasing others including Pgc1α ( D ), Pgc1β ( E ), Acox1 ( F ), and Cidea ( G ) in HIB-1B cells. TSA has no effects on Cox1 ( H ) and Cpt1b ( I ) expression. Cells were pre-treated with TSA (500 nM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Brown adipocyte mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 4–6; *p

    Techniques Used: Expressing, Quantitative RT-PCR

    Pan HDAC inhibitor TSA or class II HDAC inhibitor MC-1568 increases the expression of Rb in brown adipocytes. HIB-1B or BAT1 cells were pre-treated with TSA (500 nM) or MC-1568 (MC, 10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours (HIB-1B) or isoproterenol (ISO, 1 µM) for 3 hours (BAT1). Rb mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; *p
    Figure Legend Snippet: Pan HDAC inhibitor TSA or class II HDAC inhibitor MC-1568 increases the expression of Rb in brown adipocytes. HIB-1B or BAT1 cells were pre-treated with TSA (500 nM) or MC-1568 (MC, 10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours (HIB-1B) or isoproterenol (ISO, 1 µM) for 3 hours (BAT1). Rb mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; *p

    Techniques Used: Expressing, Quantitative RT-PCR

    5) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    6) Product Images from "HDAC inhibitors enhance the immunotherapy response of melanoma cells"

    Article Title: HDAC inhibitors enhance the immunotherapy response of melanoma cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17950

    Multiplex data comparing vehicle control to HDAC inhibitor in the presence or absence of an anti-PD-1 antibody Plasma from animals treated with: vehicle control IgG; vehicle control anti-PD-1; AR42 IgG; AR42 anti-PD-1; sodium valproate IgG; sodium valproate anti-PD-1; was processed in the MAGPIX multiplex machine to determine the concentrations of the indicated cytokines (n = 3 separate tumors +/-SEM) * p
    Figure Legend Snippet: Multiplex data comparing vehicle control to HDAC inhibitor in the presence or absence of an anti-PD-1 antibody Plasma from animals treated with: vehicle control IgG; vehicle control anti-PD-1; AR42 IgG; AR42 anti-PD-1; sodium valproate IgG; sodium valproate anti-PD-1; was processed in the MAGPIX multiplex machine to determine the concentrations of the indicated cytokines (n = 3 separate tumors +/-SEM) * p

    Techniques Used: Multiplex Assay

    Exposure of melanoma cells to [pazopanib + HDAC inhibitor] reduces the protein levels of HDACs in a Beclin1-dependent fashion (A) TPF-12-293 cells were treated with vehicle control or with [pazopanib (1 μM) + AR42 (600 nM)] for 6h. Cells were fixed in place and immunostaining at 60X magnification performed to detect the proteins indicated in each image; HDAC6, p62, LAMP2, and P-ATG13 S318. (B) TPF-12-293 cells were transfected with a scrambled control or with an siRNA to knock down Beclin1. Twenty-four h after transfection cells were treated with vehicle control or with [pazopanib (1 μM) + AR42 (600 nM)] or with [pazopanib (1 μM) + valproate (250 μM)] for 6h. Cells were fixed in place and immunostaining performed to detect the expression of HDACs1-11. The bars read from left to right: vehicle control; [PAZ+AR42]; [PAZ+VAL] (n = 3 +/-SEM). * p
    Figure Legend Snippet: Exposure of melanoma cells to [pazopanib + HDAC inhibitor] reduces the protein levels of HDACs in a Beclin1-dependent fashion (A) TPF-12-293 cells were treated with vehicle control or with [pazopanib (1 μM) + AR42 (600 nM)] for 6h. Cells were fixed in place and immunostaining at 60X magnification performed to detect the proteins indicated in each image; HDAC6, p62, LAMP2, and P-ATG13 S318. (B) TPF-12-293 cells were transfected with a scrambled control or with an siRNA to knock down Beclin1. Twenty-four h after transfection cells were treated with vehicle control or with [pazopanib (1 μM) + AR42 (600 nM)] or with [pazopanib (1 μM) + valproate (250 μM)] for 6h. Cells were fixed in place and immunostaining performed to detect the expression of HDACs1-11. The bars read from left to right: vehicle control; [PAZ+AR42]; [PAZ+VAL] (n = 3 +/-SEM). * p

    Techniques Used: Immunostaining, Transfection, Expressing

    HDAC inhibitors regulate the expression of PD-L1, PD-L2 in melanoma cells (A) Melanoma cells were treated with vehicle control or with AR42 (600 nM) for 6h. Cells were fixed in place and immunostaining performed to determine the gross (10X mag.) expression of PD-L1, PD-L2, MHCA and ODC. (n = 3 +/-SEM) *p
    Figure Legend Snippet: HDAC inhibitors regulate the expression of PD-L1, PD-L2 in melanoma cells (A) Melanoma cells were treated with vehicle control or with AR42 (600 nM) for 6h. Cells were fixed in place and immunostaining performed to determine the gross (10X mag.) expression of PD-L1, PD-L2, MHCA and ODC. (n = 3 +/-SEM) *p

    Techniques Used: Expressing, Immunostaining

    HDAC inhibitors regulate the expression of MHCA and ODC in melanoma cells (A) Melanoma cells were treated with vehicle control or with AR42 (600 nM) for 6h. Cells were fixed in place and immunostaining performed to determine the expression of MHCA and ODC (n = 3 +/-SEM). * p
    Figure Legend Snippet: HDAC inhibitors regulate the expression of MHCA and ODC in melanoma cells (A) Melanoma cells were treated with vehicle control or with AR42 (600 nM) for 6h. Cells were fixed in place and immunostaining performed to determine the expression of MHCA and ODC (n = 3 +/-SEM). * p

    Techniques Used: Expressing, Immunostaining

    7) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    8) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    9) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    10) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    11) Product Images from "Hippo signaling dysfunction induces cancer cell addiction to YAP"

    Article Title: Hippo signaling dysfunction induces cancer cell addiction to YAP

    Journal: Oncogene

    doi: 10.1038/s41388-018-0419-5

    ) ( A ) A group of HDAC inhibitors were identified to suppress YAP expression. A clinical compound library (with 146 compounds) screen was performed in MDA-MB-231 cells. YAP immunofluorescent staining was performed and its relative fluorescent intensity was calculated. Several identified hits that affect YAP fluorescent intensity were indicated. ( B ) HDAC inhibitors suppressed the YAP protein expression. Western blotting was performed with the indicated antibodies. Cells were treated for 24 hours with the indicated compounds (0.5 μM JNJ26481585, 0.5 μM LAQ824, 0.5 μM LBH589, 10 μM SAHA, 10 μM Trichostatin A, and 100 μM Nicotinamide). ( C and D ) YAP gene transcription is suppressed by HDAC inhibitors. YAP gene transcription (mean ± s.d., n = 3 biological replicates) was examined in both HEK293A ( C ) and the indicated cancer cells ( D ) after 24 hour-treatment by the indicated compounds. ** p
    Figure Legend Snippet: ) ( A ) A group of HDAC inhibitors were identified to suppress YAP expression. A clinical compound library (with 146 compounds) screen was performed in MDA-MB-231 cells. YAP immunofluorescent staining was performed and its relative fluorescent intensity was calculated. Several identified hits that affect YAP fluorescent intensity were indicated. ( B ) HDAC inhibitors suppressed the YAP protein expression. Western blotting was performed with the indicated antibodies. Cells were treated for 24 hours with the indicated compounds (0.5 μM JNJ26481585, 0.5 μM LAQ824, 0.5 μM LBH589, 10 μM SAHA, 10 μM Trichostatin A, and 100 μM Nicotinamide). ( C and D ) YAP gene transcription is suppressed by HDAC inhibitors. YAP gene transcription (mean ± s.d., n = 3 biological replicates) was examined in both HEK293A ( C ) and the indicated cancer cells ( D ) after 24 hour-treatment by the indicated compounds. ** p

    Techniques Used: Expressing, Multiple Displacement Amplification, Staining, Western Blot

    Hippo deficiency induces the tumor vulnerability to HDAC inhibitors ( A ) HDAC inhibitors suppressed Yap expression in both wild-type 4T1 and Lats1/2 DKO 4T1 cells. Western blotting was performed with the indicated antibodies. ( B and C ) Loss of Lats1/2 sensitized the 4T1-derived xenograft tumors to HDAC inhibitors. Wild-type 4T1or Lats1/2 DKO 4T1cells were subjected to the xenograft tumor assay and treated with LBH589 and SAHA. Xenograft tumors are shown in ( B ) and the tumor weight was quantified (n = 5 mice, mean ± s.d.) ( C ). * p
    Figure Legend Snippet: Hippo deficiency induces the tumor vulnerability to HDAC inhibitors ( A ) HDAC inhibitors suppressed Yap expression in both wild-type 4T1 and Lats1/2 DKO 4T1 cells. Western blotting was performed with the indicated antibodies. ( B and C ) Loss of Lats1/2 sensitized the 4T1-derived xenograft tumors to HDAC inhibitors. Wild-type 4T1or Lats1/2 DKO 4T1cells were subjected to the xenograft tumor assay and treated with LBH589 and SAHA. Xenograft tumors are shown in ( B ) and the tumor weight was quantified (n = 5 mice, mean ± s.d.) ( C ). * p

    Techniques Used: Expressing, Western Blot, Derivative Assay, Mouse Assay

    12) Product Images from "Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment"

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05178-5

    Inhibition of HDAC upregulates surface CXCR4 expression and promotes homing of human hematopoietic stem and progenitor cells. a Mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells after treating cells for 16 h with compounds (1 μM) from the SCREEN-WELL Epigenetics Library. One compound, BML-266, was excluded from the results because of unspecific autofluorescence. b Histogram of surface CXCR4 expression of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. Representative histogram from five independent experiments is shown. c Quantification of mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. None indicates the group without any treatment. Data pooled from five independent experiments are shown ( n = 5, one-way ANOVA, *** p
    Figure Legend Snippet: Inhibition of HDAC upregulates surface CXCR4 expression and promotes homing of human hematopoietic stem and progenitor cells. a Mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells after treating cells for 16 h with compounds (1 μM) from the SCREEN-WELL Epigenetics Library. One compound, BML-266, was excluded from the results because of unspecific autofluorescence. b Histogram of surface CXCR4 expression of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. Representative histogram from five independent experiments is shown. c Quantification of mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. None indicates the group without any treatment. Data pooled from five independent experiments are shown ( n = 5, one-way ANOVA, *** p

    Techniques Used: Inhibition, Expressing, Fluorescence

    Inhibition of HDAC promotes long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or M344 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or M344. The data were pooled from two independent experiments ( n = 10 mice for vehicle group, n = 9 for M344 group, t -test, ** p
    Figure Legend Snippet: Inhibition of HDAC promotes long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or M344 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or M344. The data were pooled from two independent experiments ( n = 10 mice for vehicle group, n = 9 for M344 group, t -test, ** p

    Techniques Used: Inhibition, Flow Cytometry, Mouse Assay, Transplantation Assay, Negative Control

    13) Product Images from "Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment"

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05178-5

    Inhibition of HDAC upregulates surface CXCR4 expression and promotes homing of human hematopoietic stem and progenitor cells. a Mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells after treating cells for 16 h with compounds (1 μM) from the SCREEN-WELL Epigenetics Library. One compound, BML-266, was excluded from the results because of unspecific autofluorescence. b Histogram of surface CXCR4 expression of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. Representative histogram from five independent experiments is shown. c Quantification of mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. None indicates the group without any treatment. Data pooled from five independent experiments are shown ( n = 5, one-way ANOVA, *** p
    Figure Legend Snippet: Inhibition of HDAC upregulates surface CXCR4 expression and promotes homing of human hematopoietic stem and progenitor cells. a Mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells after treating cells for 16 h with compounds (1 μM) from the SCREEN-WELL Epigenetics Library. One compound, BML-266, was excluded from the results because of unspecific autofluorescence. b Histogram of surface CXCR4 expression of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. Representative histogram from five independent experiments is shown. c Quantification of mean fluorescence intensity (MFI) of surface CXCR4 of human CB CD34 + cells treated with vehicle or HDAC inhibitor M344. None indicates the group without any treatment. Data pooled from five independent experiments are shown ( n = 5, one-way ANOVA, *** p

    Techniques Used: Inhibition, Expressing, Fluorescence

    Inhibition of HDAC promotes long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or M344 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or M344. The data were pooled from two independent experiments ( n = 10 mice for vehicle group, n = 9 for M344 group, t -test, ** p
    Figure Legend Snippet: Inhibition of HDAC promotes long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or M344 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or M344. The data were pooled from two independent experiments ( n = 10 mice for vehicle group, n = 9 for M344 group, t -test, ** p

    Techniques Used: Inhibition, Flow Cytometry, Mouse Assay, Transplantation Assay, Negative Control

    14) Product Images from "Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6"

    Article Title: Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00724.2016

    A–D : HAEC were incubated under control conditions (Con) or with the broad-spectrum HDAC inhibitors trichostatin A (TSA, 0.5 μM), Scriptaid (SCR, 0.5 μM), and suberoylanilide hydroxamic acid (SAHA, 0.5 μM) or the class III-specific HDAC inhibitor nicotinamide (NAM, 5 μM); MS-275 (1 μM, an inhibitor of class I and III HDACs) or MGCD0103 (MGCD, 1 μM, an inhibitor of class I HDACs); increasing doses of TMP269 (TMP, 3 and 10 µM); or the HDAC6-specific inhibitor tubacin (1 and 10 μM). Cell lysates were subjected to Western blotting using CSEγ and GAPDH antibodies. E : densitometric analyses of data from replicate blots from conditions in C and D . F : CSE γ promoter activity for the fragment 1 kb upstream of the transcription start site was measured using a luciferase reporter assay in HAEC transduced with adenoviruses encoding firefly luciferase controlled by the 1-kb CSEγ promoter in the presence or absence of tubacin (10 μM). Adenoviruses encoding Renilla luciferase controlled by a ubiquitous receptor tyrosine kinase (RTK) promoter were used for normalization. G and H : HAEC incubated with or without OxLDL (50 μg/ml) were subjected to Western blotting using HDAC6, acetylated (Ac) α-tubulin, and total α-tubulin antibodies. I : real-time PCR using HDAC6- and 18s-specific primers. J : HAEC were cultured in the absence or presence of OxLDL (25 and 50 μg/ml) for 24 h, and cell lysates were immunoblotted with HDAC2 or GAPDH antibodies. *, # P
    Figure Legend Snippet: A–D : HAEC were incubated under control conditions (Con) or with the broad-spectrum HDAC inhibitors trichostatin A (TSA, 0.5 μM), Scriptaid (SCR, 0.5 μM), and suberoylanilide hydroxamic acid (SAHA, 0.5 μM) or the class III-specific HDAC inhibitor nicotinamide (NAM, 5 μM); MS-275 (1 μM, an inhibitor of class I and III HDACs) or MGCD0103 (MGCD, 1 μM, an inhibitor of class I HDACs); increasing doses of TMP269 (TMP, 3 and 10 µM); or the HDAC6-specific inhibitor tubacin (1 and 10 μM). Cell lysates were subjected to Western blotting using CSEγ and GAPDH antibodies. E : densitometric analyses of data from replicate blots from conditions in C and D . F : CSE γ promoter activity for the fragment 1 kb upstream of the transcription start site was measured using a luciferase reporter assay in HAEC transduced with adenoviruses encoding firefly luciferase controlled by the 1-kb CSEγ promoter in the presence or absence of tubacin (10 μM). Adenoviruses encoding Renilla luciferase controlled by a ubiquitous receptor tyrosine kinase (RTK) promoter were used for normalization. G and H : HAEC incubated with or without OxLDL (50 μg/ml) were subjected to Western blotting using HDAC6, acetylated (Ac) α-tubulin, and total α-tubulin antibodies. I : real-time PCR using HDAC6- and 18s-specific primers. J : HAEC were cultured in the absence or presence of OxLDL (25 and 50 μg/ml) for 24 h, and cell lysates were immunoblotted with HDAC2 or GAPDH antibodies. *, # P

    Techniques Used: Incubation, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transduction, Real-time Polymerase Chain Reaction, Cell Culture

    15) Product Images from "Hippo signaling dysfunction induces cancer cell addiction to YAP"

    Article Title: Hippo signaling dysfunction induces cancer cell addiction to YAP

    Journal: Oncogene

    doi: 10.1038/s41388-018-0419-5

    HDAC inhibitors suppress the YAP gene expression and the YAP-dependent cell viability. (See also Figure S2 ) ( A ) A group of HDAC inhibitors were identified to suppress YAP expression. A clinical compound library (with 146 compounds) screen was performed in MDA-MB-231 cells. YAP immunofluorescent staining was performed and its relative fluorescent intensity was calculated. Several identified hits that affect YAP fluorescent intensity were indicated. ( B ) HDAC inhibitors suppressed the YAP protein expression. Western blotting was performed with the indicated antibodies. Cells were treated for 24 hours with the indicated compounds (0.5 μM JNJ26481585, 0.5 μM LAQ824, 0.5 μM LBH589, 10 μM SAHA, 10 μM Trichostatin A, and 100 μM Nicotinamide). ( C and D ) YAP gene transcription is suppressed by HDAC inhibitors. YAP gene transcription (mean ± s.d., n = 3 biological replicates) was examined in both HEK293A ( C ) and the indicated cancer cells ( D ) after 24 hour-treatment by the indicated compounds. ** p
    Figure Legend Snippet: HDAC inhibitors suppress the YAP gene expression and the YAP-dependent cell viability. (See also Figure S2 ) ( A ) A group of HDAC inhibitors were identified to suppress YAP expression. A clinical compound library (with 146 compounds) screen was performed in MDA-MB-231 cells. YAP immunofluorescent staining was performed and its relative fluorescent intensity was calculated. Several identified hits that affect YAP fluorescent intensity were indicated. ( B ) HDAC inhibitors suppressed the YAP protein expression. Western blotting was performed with the indicated antibodies. Cells were treated for 24 hours with the indicated compounds (0.5 μM JNJ26481585, 0.5 μM LAQ824, 0.5 μM LBH589, 10 μM SAHA, 10 μM Trichostatin A, and 100 μM Nicotinamide). ( C and D ) YAP gene transcription is suppressed by HDAC inhibitors. YAP gene transcription (mean ± s.d., n = 3 biological replicates) was examined in both HEK293A ( C ) and the indicated cancer cells ( D ) after 24 hour-treatment by the indicated compounds. ** p

    Techniques Used: Expressing, Multiple Displacement Amplification, Staining, Western Blot

    Hippo deficiency induces the tumor vulnerability to HDAC inhibitors ( A ) HDAC inhibitors suppressed Yap expression in both wild-type 4T1 and Lats1/2 DKO 4T1 cells. Western blotting was performed with the indicated antibodies. ( B and C ) Loss of Lats1/2 sensitized the 4T1-derived xenograft tumors to HDAC inhibitors. Wild-type 4T1or Lats1/2 DKO 4T1cells were subjected to the xenograft tumor assay and treated with LBH589 and SAHA. Xenograft tumors are shown in ( B ) and the tumor weight was quantified (n = 5 mice, mean ± s.d.) ( C ). * p
    Figure Legend Snippet: Hippo deficiency induces the tumor vulnerability to HDAC inhibitors ( A ) HDAC inhibitors suppressed Yap expression in both wild-type 4T1 and Lats1/2 DKO 4T1 cells. Western blotting was performed with the indicated antibodies. ( B and C ) Loss of Lats1/2 sensitized the 4T1-derived xenograft tumors to HDAC inhibitors. Wild-type 4T1or Lats1/2 DKO 4T1cells were subjected to the xenograft tumor assay and treated with LBH589 and SAHA. Xenograft tumors are shown in ( B ) and the tumor weight was quantified (n = 5 mice, mean ± s.d.) ( C ). * p

    Techniques Used: Expressing, Western Blot, Derivative Assay, Mouse Assay

    16) Product Images from "Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors"

    Article Title: Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

    Journal: Oncotarget

    doi:

    Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).
    Figure Legend Snippet: Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).

    Techniques Used: Activity Assay, Transfection, Expressing, Glo Assay

    BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).
    Figure Legend Snippet: BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).

    Techniques Used: Glo Assay, Transfection, Expressing

    17) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    18) Product Images from "Class I and II Histone Deacetylase Inhibitors Differentially Regulate Thermogenic Gene Expression in Brown Adipocytes"

    Article Title: Class I and II Histone Deacetylase Inhibitors Differentially Regulate Thermogenic Gene Expression in Brown Adipocytes

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31560-w

    Class II HDAC inhibitor MC-1568 inhibits the expression of Ucp1 , Pparγ and Prdm16 , but stimulates the expression of Pgc1α , Pgc1β , Acox1 and Cidea in brown adipocytes. HIB-1B cells were pre-treated with MC-1568 (10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Gene expression was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; Bars with different letters are significantly different from each other in control groups (without NE) or NE-treated groups.
    Figure Legend Snippet: Class II HDAC inhibitor MC-1568 inhibits the expression of Ucp1 , Pparγ and Prdm16 , but stimulates the expression of Pgc1α , Pgc1β , Acox1 and Cidea in brown adipocytes. HIB-1B cells were pre-treated with MC-1568 (10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Gene expression was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; Bars with different letters are significantly different from each other in control groups (without NE) or NE-treated groups.

    Techniques Used: Expressing, Quantitative RT-PCR

    Pan-HDAC inhibitor TSA exhibits a mixed effect on the expression of brown fat genes. TSA inhibits the expression of brown fat genes Ucp1 ( A ), Pparγ ( B ), and Prdm16 ( C ), while increasing others including Pgc1α ( D ), Pgc1β ( E ), Acox1 ( F ), and Cidea ( G ) in HIB-1B cells. TSA has no effects on Cox1 ( H ) and Cpt1b ( I ) expression. Cells were pre-treated with TSA (500 nM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Brown adipocyte mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 4–6; *p
    Figure Legend Snippet: Pan-HDAC inhibitor TSA exhibits a mixed effect on the expression of brown fat genes. TSA inhibits the expression of brown fat genes Ucp1 ( A ), Pparγ ( B ), and Prdm16 ( C ), while increasing others including Pgc1α ( D ), Pgc1β ( E ), Acox1 ( F ), and Cidea ( G ) in HIB-1B cells. TSA has no effects on Cox1 ( H ) and Cpt1b ( I ) expression. Cells were pre-treated with TSA (500 nM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours. Brown adipocyte mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 4–6; *p

    Techniques Used: Expressing, Quantitative RT-PCR

    Pan HDAC inhibitor TSA or class II HDAC inhibitor MC-1568 increases the expression of Rb in brown adipocytes. HIB-1B or BAT1 cells were pre-treated with TSA (500 nM) or MC-1568 (MC, 10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours (HIB-1B) or isoproterenol (ISO, 1 µM) for 3 hours (BAT1). Rb mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; *p
    Figure Legend Snippet: Pan HDAC inhibitor TSA or class II HDAC inhibitor MC-1568 increases the expression of Rb in brown adipocytes. HIB-1B or BAT1 cells were pre-treated with TSA (500 nM) or MC-1568 (MC, 10 or 30 µM) for 30 min, followed by stimulation with norepinephrine (NE, 1 µM) for 4 hours (HIB-1B) or isoproterenol (ISO, 1 µM) for 3 hours (BAT1). Rb mRNA was measured by quantitative RT-PCR as described in Methods. All data are expressed as mean ± SEM, n = 3–6; *p

    Techniques Used: Expressing, Quantitative RT-PCR

    19) Product Images from "HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent"

    Article Title: HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent

    Journal: Journal of Interferon & Cytokine Research

    doi: 10.1089/jir.2014.0022

    HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat
    Figure Legend Snippet: HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat

    Techniques Used: Inhibition

    The class-I selective HDAC inhibitor MS-275 does not affect cytokine-induced Tyr701 STAT1 phosphorylation. (A) INS-1E cells were either preincubated (1 h) with MS-275 (1 μM) or coincubated (marked with*) with cytokines (IL-1β,
    Figure Legend Snippet: The class-I selective HDAC inhibitor MS-275 does not affect cytokine-induced Tyr701 STAT1 phosphorylation. (A) INS-1E cells were either preincubated (1 h) with MS-275 (1 μM) or coincubated (marked with*) with cytokines (IL-1β,

    Techniques Used: Mass Spectrometry

    Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min
    Figure Legend Snippet: Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min

    Techniques Used: Inhibition

    20) Product Images from "HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent"

    Article Title: HDAC Inhibitor-Mediated Beta-Cell Protection Against Cytokine-Induced Toxicity Is STAT1 Tyr701 Phosphorylation Independent

    Journal: Journal of Interferon & Cytokine Research

    doi: 10.1089/jir.2014.0022

    HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat
    Figure Legend Snippet: HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation or STAT1 acetylation, but knockdown of HDAC1 increases IFN-γ-induced Tyr701 STAT1 phosphorylation. (A) INS-1 cells were preincubated (24 h) with givinostat

    Techniques Used: Inhibition

    The class-I selective HDAC inhibitor MS-275 does not affect cytokine-induced Tyr701 STAT1 phosphorylation. (A) INS-1E cells were either preincubated (1 h) with MS-275 (1 μM) or coincubated (marked with*) with cytokines (IL-1β,
    Figure Legend Snippet: The class-I selective HDAC inhibitor MS-275 does not affect cytokine-induced Tyr701 STAT1 phosphorylation. (A) INS-1E cells were either preincubated (1 h) with MS-275 (1 μM) or coincubated (marked with*) with cytokines (IL-1β,

    Techniques Used: Mass Spectrometry

    Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min
    Figure Legend Snippet: Nonselective HDAC inhibition does not affect IFN-γ-induced Tyr701 STAT1 phosphorylation. INS-1 cells were preincubated (1 h) with givinostat (125 nM) before exposure to IFN-γ (1.3 ng/mL) for 15 or 30 min

    Techniques Used: Inhibition

    21) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    22) Product Images from "Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways"

    Article Title: Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1β-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3410

    Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.
    Figure Legend Snippet: Possible signaling pathways associated with ADAMTS, col X, and COX-2 regulation by IL-1β in chondrocytes. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; col X, type X collagen; COX2, cyclooxygenase; IL, interleukin; HDAC, histone deacetylase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; TSA, Trichostatin A; PCI, PCI-34051.

    Techniques Used: Histone Deacetylase Assay

    HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: HDAC regulates the osteoarthritis-related genes and protein expression of rACs via ERK1/2 and JNK. (A) IL-1β-induced ERK1/2 phosphorylation was blocked by TSA, and not blocked by PCI; JNK phosphorylation was blocked by TSA and PCI. (B) HDAC inhibitors (TSA or PCI) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related genes (col X, COX2, ADAMTS-5 and ADAMTS-4) expression induced by IL-1β. Results are presented with respect to GAPDH expression. (C) siRNAs (targeting HDAC4 or HDAC8) suppressed HDAC expression at both mRNA and protein level, and prevented the upregulation of osteoarthritis-related gene expression induced by IL-1β. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Standard Deviation

    The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P
    Figure Legend Snippet: The effect of HDAC inhibitors on ADAMTS-4 and ADAMTS-5 in IL-1β-treated rACs. (A) HDAC 1–11 mRNA expression in rACs was determined by reverse transcription-quantitative polymerase chain reaction. HDAC 4 and HDAC 8 had significantly higher expression in IL-1β-treated group compared with the control group. Results are expressed with respect to GAPDH expression. Data represent the means ± standard deviation, n=3. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

    23) Product Images from "Identification of HDAC inhibitors using a cell-based HDAC I/II assay"

    Article Title: Identification of HDAC inhibitors using a cell-based HDAC I/II assay

    Journal: Journal of biomolecular screening

    doi: 10.1177/1087057116629381

    Optimization and validation of the histone deacetylase (HDAC)-Glo I/II assay in 1536-well format
    Figure Legend Snippet: Optimization and validation of the histone deacetylase (HDAC)-Glo I/II assay in 1536-well format

    Techniques Used: Histone Deacetylase Assay, Ii Assay

    24) Product Images from "Isoform-specific characterization of class I histone deacetylases and their therapeutic modulation in pulmonary hypertension"

    Article Title: Isoform-specific characterization of class I histone deacetylases and their therapeutic modulation in pulmonary hypertension

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69737-x

    Transcriptome and pathways regulated by HDAC isoforms in PAH. RNA-interference was achieved by transient transfection of IPAH-PAAFs cultured ex vivo with validated HDAC1, HDAC2 and HDAC8 siRNAs. ( A ) qPCR analysis was performed on the RNA isolated from IPAH-PAAFs (n = 3), 24 h post-transfection. ∆C t values were calculated using β2M as reference and further normalized (∆∆C t ) to the respective solvent concentrations (DMSO or water). ( B ) Functional effects of RNA-interference on fetal calf serum (FCS) induced IPAH-PAAF proliferation was assessed by BrdU incorporation, 48 h post-transfection. Similarly, ( C ) functional effects of RNA-interference on serum starvation (0.1% FCS) induced apoptosis in IPAH-PAAFs was assessed by Cell Death Detection ELISA PLUS , 48 h post-transfection. Data was normalized to untransfected cells and visualized (IPAH; n = 3; *p
    Figure Legend Snippet: Transcriptome and pathways regulated by HDAC isoforms in PAH. RNA-interference was achieved by transient transfection of IPAH-PAAFs cultured ex vivo with validated HDAC1, HDAC2 and HDAC8 siRNAs. ( A ) qPCR analysis was performed on the RNA isolated from IPAH-PAAFs (n = 3), 24 h post-transfection. ∆C t values were calculated using β2M as reference and further normalized (∆∆C t ) to the respective solvent concentrations (DMSO or water). ( B ) Functional effects of RNA-interference on fetal calf serum (FCS) induced IPAH-PAAF proliferation was assessed by BrdU incorporation, 48 h post-transfection. Similarly, ( C ) functional effects of RNA-interference on serum starvation (0.1% FCS) induced apoptosis in IPAH-PAAFs was assessed by Cell Death Detection ELISA PLUS , 48 h post-transfection. Data was normalized to untransfected cells and visualized (IPAH; n = 3; *p

    Techniques Used: Transfection, Cell Culture, Ex Vivo, Real-time Polymerase Chain Reaction, Isolation, Functional Assay, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay

    Immunolocalization of class I HDAC isoforms in human lungs from donors and IPAH patients. Representative microscopic pictures of human pulmonary arteries immunostained for ( A ) HDAC1, ( B ) HDAC2 and ( C ) HDAC8 expression in human donor and IPAH lung sections. HDAC (in green fluorescence) expression was localized to different vascular by staining for cell-specific markers such as von Willebrand factor (vWF, Red) for endothelial cells, α-smooth muscle actin (SMA or ACTA2, Red) for smooth muscle cells and collagen I (Col1 or COL1A1, Red) associated with adventitial fibroblasts, while the nucleus was counter-stained with DAPI (Blue). Scale bar: 50 µm.
    Figure Legend Snippet: Immunolocalization of class I HDAC isoforms in human lungs from donors and IPAH patients. Representative microscopic pictures of human pulmonary arteries immunostained for ( A ) HDAC1, ( B ) HDAC2 and ( C ) HDAC8 expression in human donor and IPAH lung sections. HDAC (in green fluorescence) expression was localized to different vascular by staining for cell-specific markers such as von Willebrand factor (vWF, Red) for endothelial cells, α-smooth muscle actin (SMA or ACTA2, Red) for smooth muscle cells and collagen I (Col1 or COL1A1, Red) associated with adventitial fibroblasts, while the nucleus was counter-stained with DAPI (Blue). Scale bar: 50 µm.

    Techniques Used: Expressing, Fluorescence, Staining

    Evaluation of isoform-selective HDAC inhibitors in chronic hypoxia-induced PH and RV hypertrophy in vivo. ( A ) Rats were divided into seven groups and exposed to, (1) normoxia (N), (2) hypoxia (H) for 4 weeks, and hypoxia plus (3) CAY10398, (4) PCI34051 (P), (5) VPA (V), (6) SAHA (S) and (7) Romidepsin (R). Compounds were administered during the last 2 weeks of 4 week hypoxia exposure (n = 6 rats were assigned per group treated with isoform-selective inhibitors). At the end of experiment, haemodynamic parameters were measured and analysed in the animals that completed the treatment regimen. The parameters analysed were ( B ) mean pulmonary artery pressure (mPAP), ( C ) right ventricular systolic pressure (RVSP), ( D ) systolic blood pressure, (SBP), ( E ) right ventricular hypertrophy (RV/LV + septum), and ( F ) percentage of muscularised vessels. Data are represented as bar charts (n ≥ 3; *p
    Figure Legend Snippet: Evaluation of isoform-selective HDAC inhibitors in chronic hypoxia-induced PH and RV hypertrophy in vivo. ( A ) Rats were divided into seven groups and exposed to, (1) normoxia (N), (2) hypoxia (H) for 4 weeks, and hypoxia plus (3) CAY10398, (4) PCI34051 (P), (5) VPA (V), (6) SAHA (S) and (7) Romidepsin (R). Compounds were administered during the last 2 weeks of 4 week hypoxia exposure (n = 6 rats were assigned per group treated with isoform-selective inhibitors). At the end of experiment, haemodynamic parameters were measured and analysed in the animals that completed the treatment regimen. The parameters analysed were ( B ) mean pulmonary artery pressure (mPAP), ( C ) right ventricular systolic pressure (RVSP), ( D ) systolic blood pressure, (SBP), ( E ) right ventricular hypertrophy (RV/LV + septum), and ( F ) percentage of muscularised vessels. Data are represented as bar charts (n ≥ 3; *p

    Techniques Used: In Vivo

    Class I HDAC isoforms are overexpressed in PAH and are associated with adventitial fibroblast proliferation. Cell proliferation was assessed by BrdU incorporation in ( A ) donor- and IPAH-PAAFs (n = 3) cultured ex vivo. Data was normalized to respective donors. ( B ) qPCR analysis of HDACs was performed on the RNA isolated from the PAAFs harvested from healthy donors (n = 4) and PAH (n = 4) patients. ∆C t values were normalized to β-2 microglobulin (β2M) and further normalized (∆∆C t ) to donor controls. ( C ) Western blots were performed on lysates extracted from human PAAFs of healthy donors (n = 4) and PAH (n = 4) patients. ( D ) Blots were quantified by densitometry and are represented as box plots after normalization to internal loading control. ( E ) HDAC activity (OD/min/mg) was measured in PAAFs harvested from healthy donors (n = 3) and PAH (n = 3) patients using colorimetric assay kit (Epigenase HDAC Activity/Inhibition Assay Kit, Epigentek). ( F ) Overexpression of HDACs was achieved by transient transfection of donor-PAAFs (n = 3) with validated HDAC1, HDAC2, HDAC8 plasmids, and their functional effects on proliferation was assessed by BrdU incorporation, 48 hours (h) post-transfection. Empty vector plasmid was used as negative control. Data was normalized to untransfected cells. In all plots, significant differences between corresponding comparisons are indicated as *p
    Figure Legend Snippet: Class I HDAC isoforms are overexpressed in PAH and are associated with adventitial fibroblast proliferation. Cell proliferation was assessed by BrdU incorporation in ( A ) donor- and IPAH-PAAFs (n = 3) cultured ex vivo. Data was normalized to respective donors. ( B ) qPCR analysis of HDACs was performed on the RNA isolated from the PAAFs harvested from healthy donors (n = 4) and PAH (n = 4) patients. ∆C t values were normalized to β-2 microglobulin (β2M) and further normalized (∆∆C t ) to donor controls. ( C ) Western blots were performed on lysates extracted from human PAAFs of healthy donors (n = 4) and PAH (n = 4) patients. ( D ) Blots were quantified by densitometry and are represented as box plots after normalization to internal loading control. ( E ) HDAC activity (OD/min/mg) was measured in PAAFs harvested from healthy donors (n = 3) and PAH (n = 3) patients using colorimetric assay kit (Epigenase HDAC Activity/Inhibition Assay Kit, Epigentek). ( F ) Overexpression of HDACs was achieved by transient transfection of donor-PAAFs (n = 3) with validated HDAC1, HDAC2, HDAC8 plasmids, and their functional effects on proliferation was assessed by BrdU incorporation, 48 hours (h) post-transfection. Empty vector plasmid was used as negative control. Data was normalized to untransfected cells. In all plots, significant differences between corresponding comparisons are indicated as *p

    Techniques Used: BrdU Incorporation Assay, Cell Culture, Ex Vivo, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Activity Assay, Colorimetric Assay, Inhibition, Over Expression, Transfection, Functional Assay, Plasmid Preparation, Negative Control

    Isoform-selective HDAC activity inhibition reverses hypertensive phenotypes in PAH fibroblasts ex vivo. Pharmacological HDAC inhibition suppresses hyper-proliferative phenotype and reverses resistance to apoptosis in IPAH-PAAFs ex vivo. IPAH-PAAFs were treated with increasing concentrations of commercially available ( A ) pan-HDAC inhibitor Vorinostat (SAHA), ( B ) class-selective Valproic acid (VPA) and isoform selective inhibitors such as ( C ) CAY10398, ( D ) Romidepsin, ( E ) PCI34051 or their respective solvents (DMSO or water). Cell proliferation was assessed by BrdU incorporation and induction of apoptosis was assessed by Cell Death Detection ELISA PLUS , 24 h post-treatment. Absorbance values obtained for HDAC inhibitor and solvent treatments were normalized to the BrdU incorporation of untreated cells. Data are represented as mean ± SEM (n = 3; *p
    Figure Legend Snippet: Isoform-selective HDAC activity inhibition reverses hypertensive phenotypes in PAH fibroblasts ex vivo. Pharmacological HDAC inhibition suppresses hyper-proliferative phenotype and reverses resistance to apoptosis in IPAH-PAAFs ex vivo. IPAH-PAAFs were treated with increasing concentrations of commercially available ( A ) pan-HDAC inhibitor Vorinostat (SAHA), ( B ) class-selective Valproic acid (VPA) and isoform selective inhibitors such as ( C ) CAY10398, ( D ) Romidepsin, ( E ) PCI34051 or their respective solvents (DMSO or water). Cell proliferation was assessed by BrdU incorporation and induction of apoptosis was assessed by Cell Death Detection ELISA PLUS , 24 h post-treatment. Absorbance values obtained for HDAC inhibitor and solvent treatments were normalized to the BrdU incorporation of untreated cells. Data are represented as mean ± SEM (n = 3; *p

    Techniques Used: Activity Assay, Inhibition, Ex Vivo, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay

    Expression of class I HDAC isoforms are altered in different cardiopulmonary tissues from human PAH. ( A ) Schematic representation of the mammalian class I HDACs. ( B ) Real-time quantitative polymerase chain reaction (qPCR) analysis was performed with primer pairs (Supplementary Table 1) on the complementary DNA (cDNA) synthesized from the RNA isolated from human lung homogenates of healthy donors (n = 5) and IPAH (n = 5) patients. All values were normalized against hypoxanthine phosphoribosyltransferase (HPRT). ( C ) Western blot was performed on human lung homogenates from donors (n = 6) and PAH (n = 7) patients using validated antibodies listed in Supplementary Table 2. ( D ) Blots from lung homogenates were quantified by densitometry and are represented as box plots after normalization to internal loading control. ( E ) The transcription levels of HDAC isoforms were quantified using qPCR on the total RNA isolated from the laser micro-dissected intrapulmonary arteries ( > 50 µm and
    Figure Legend Snippet: Expression of class I HDAC isoforms are altered in different cardiopulmonary tissues from human PAH. ( A ) Schematic representation of the mammalian class I HDACs. ( B ) Real-time quantitative polymerase chain reaction (qPCR) analysis was performed with primer pairs (Supplementary Table 1) on the complementary DNA (cDNA) synthesized from the RNA isolated from human lung homogenates of healthy donors (n = 5) and IPAH (n = 5) patients. All values were normalized against hypoxanthine phosphoribosyltransferase (HPRT). ( C ) Western blot was performed on human lung homogenates from donors (n = 6) and PAH (n = 7) patients using validated antibodies listed in Supplementary Table 2. ( D ) Blots from lung homogenates were quantified by densitometry and are represented as box plots after normalization to internal loading control. ( E ) The transcription levels of HDAC isoforms were quantified using qPCR on the total RNA isolated from the laser micro-dissected intrapulmonary arteries ( > 50 µm and

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Synthesized, Isolation, Western Blot

    Regulation of class I HDACs and histone modifications by hypoxia ex vivo. ( A ) Schematic representation of the experimental plan to study the regulation of HDAC expression and dynamic changes in histone acetylation levels in donor-PAAFs (n ≥ 3) exposed to hypoxia. ( B ) Western blots were performed on proteins extracted from human donor-PAAFs exposed to hypoxia. ( C ) Blots were quantified by densitometry and are represented as bar charts after normalization to internal loading control, β-actin. For HDAC8, both the upper (Fig. 6) and lower bands (in Fig. S3) were quantified separately. ( D ) To determine the effect of hypoxia exposure on histone modifications, western blot analysis was performed on extracts from donor-PAAFs exposed to hypoxia with antibodies raised against specific post-translational modification of histones associated with transcription activation (H3K4ac; H3K9/K14ac) and active enhancers (H3K27ac). ( E ) Blots were quantified by densitometric analysis and are represented as bar charts after normalization to internal loading control (Pan-histone H3). Significant differences found in comparison between the treatment (hypoxia) and control groups (normoxia) are indicated by an asterisk symbol (*p
    Figure Legend Snippet: Regulation of class I HDACs and histone modifications by hypoxia ex vivo. ( A ) Schematic representation of the experimental plan to study the regulation of HDAC expression and dynamic changes in histone acetylation levels in donor-PAAFs (n ≥ 3) exposed to hypoxia. ( B ) Western blots were performed on proteins extracted from human donor-PAAFs exposed to hypoxia. ( C ) Blots were quantified by densitometry and are represented as bar charts after normalization to internal loading control, β-actin. For HDAC8, both the upper (Fig. 6) and lower bands (in Fig. S3) were quantified separately. ( D ) To determine the effect of hypoxia exposure on histone modifications, western blot analysis was performed on extracts from donor-PAAFs exposed to hypoxia with antibodies raised against specific post-translational modification of histones associated with transcription activation (H3K4ac; H3K9/K14ac) and active enhancers (H3K27ac). ( E ) Blots were quantified by densitometric analysis and are represented as bar charts after normalization to internal loading control (Pan-histone H3). Significant differences found in comparison between the treatment (hypoxia) and control groups (normoxia) are indicated by an asterisk symbol (*p

    Techniques Used: Ex Vivo, Expressing, Western Blot, Modification, Activation Assay

    25) Product Images from "Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6"

    Article Title: Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00724.2016

    A–D : HAEC were incubated under control conditions (Con) or with the broad-spectrum HDAC inhibitors trichostatin A (TSA, 0.5 μM), Scriptaid (SCR, 0.5 μM), and suberoylanilide hydroxamic acid (SAHA, 0.5 μM) or the class III-specific HDAC inhibitor nicotinamide (NAM, 5 μM); MS-275 (1 μM, an inhibitor of class I and III HDACs) or MGCD0103 (MGCD, 1 μM, an inhibitor of class I HDACs); increasing doses of TMP269 (TMP, 3 and 10 µM); or the HDAC6-specific inhibitor tubacin (1 and 10 μM). Cell lysates were subjected to Western blotting using CSEγ and GAPDH antibodies. E : densitometric analyses of data from replicate blots from conditions in C and D . F : CSE γ promoter activity for the fragment 1 kb upstream of the transcription start site was measured using a luciferase reporter assay in HAEC transduced with adenoviruses encoding firefly luciferase controlled by the 1-kb CSEγ promoter in the presence or absence of tubacin (10 μM). Adenoviruses encoding Renilla luciferase controlled by a ubiquitous receptor tyrosine kinase (RTK) promoter were used for normalization. G and H : HAEC incubated with or without OxLDL (50 μg/ml) were subjected to Western blotting using HDAC6, acetylated (Ac) α-tubulin, and total α-tubulin antibodies. I : real-time PCR using HDAC6- and 18s-specific primers. J : HAEC were cultured in the absence or presence of OxLDL (25 and 50 μg/ml) for 24 h, and cell lysates were immunoblotted with HDAC2 or GAPDH antibodies. *, # P
    Figure Legend Snippet: A–D : HAEC were incubated under control conditions (Con) or with the broad-spectrum HDAC inhibitors trichostatin A (TSA, 0.5 μM), Scriptaid (SCR, 0.5 μM), and suberoylanilide hydroxamic acid (SAHA, 0.5 μM) or the class III-specific HDAC inhibitor nicotinamide (NAM, 5 μM); MS-275 (1 μM, an inhibitor of class I and III HDACs) or MGCD0103 (MGCD, 1 μM, an inhibitor of class I HDACs); increasing doses of TMP269 (TMP, 3 and 10 µM); or the HDAC6-specific inhibitor tubacin (1 and 10 μM). Cell lysates were subjected to Western blotting using CSEγ and GAPDH antibodies. E : densitometric analyses of data from replicate blots from conditions in C and D . F : CSE γ promoter activity for the fragment 1 kb upstream of the transcription start site was measured using a luciferase reporter assay in HAEC transduced with adenoviruses encoding firefly luciferase controlled by the 1-kb CSEγ promoter in the presence or absence of tubacin (10 μM). Adenoviruses encoding Renilla luciferase controlled by a ubiquitous receptor tyrosine kinase (RTK) promoter were used for normalization. G and H : HAEC incubated with or without OxLDL (50 μg/ml) were subjected to Western blotting using HDAC6, acetylated (Ac) α-tubulin, and total α-tubulin antibodies. I : real-time PCR using HDAC6- and 18s-specific primers. J : HAEC were cultured in the absence or presence of OxLDL (25 and 50 μg/ml) for 24 h, and cell lysates were immunoblotted with HDAC2 or GAPDH antibodies. *, # P

    Techniques Used: Incubation, Mass Spectrometry, Western Blot, Activity Assay, Luciferase, Reporter Assay, Transduction, Real-time Polymerase Chain Reaction, Cell Culture

    26) Product Images from "Expression of Histone Deacetylases in Cellular Compartments of the Mouse Brain and the Effects of Ischemia"

    Article Title: Expression of Histone Deacetylases in Cellular Compartments of the Mouse Brain and the Effects of Ischemia

    Journal: Translational stroke research

    doi: 10.1007/s12975-011-0087-z

    HDAC inhibition attenuates upregulation of class I HDAC isoforms. After 60 min of OGD, levels of HDAC 1–3 expression were upregulated in all glial cell nuclei ( arrowheads ) and processes of astrocytes ( arrows ) in MON. Note that OGD upregulated
    Figure Legend Snippet: HDAC inhibition attenuates upregulation of class I HDAC isoforms. After 60 min of OGD, levels of HDAC 1–3 expression were upregulated in all glial cell nuclei ( arrowheads ) and processes of astrocytes ( arrows ) in MON. Note that OGD upregulated

    Techniques Used: Inhibition, Expressing

    27) Product Images from "Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis"

    Article Title: Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-021-02125-z

    HDAC inhibitors increase susceptibility of EwS to first line chemotherapeutics. a Heatmaps showing excess over Bliss synergy scores for combination treatment of Doxorubicin with HDACi FK228 (left) or MS-275 (right) in CHLA-10 or SK-N-MC cells. Scores greater than 1 (shown in red) denote synergistic combinations, whereas scores less than 1 (shown in green) denote antagonistic combinations. Data represent 3 technical triplicates. b Western blot analysis of caspase 3 and PARP as a measure of apoptosis susceptibility after treatment with Doxorubicin and HDACi, either in combination or individually. CHLA-10 were treated for 36 h and SK-N-MC for 24 h. Tubulin was used as loading control. c Focused heat map of genes, ≥1.5-fold differentially expressed in EwS line EW7 after combined treatment with DMSO, HDACi and/or first line chemotherapeutics Doxorubicin or Vincristine for 17 h. Treatment with Doxorubicin or Vincristine and MS-275, respectively, are highlighted in red. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162785). d GSEA enrichment plots of up- and downregulated gene sets after combined treatment with MS-275 and Doxorubicin. NES: normalized enrichment score. GSEA: http://www.broadinstitute.org/gsea/index.jsp . e GSEA leading-edge analysis of identified gene sets (C5_all, GO gene sets) downregulated after combined treatment with MS-275 and Doxorubicin. Set-to-set analysis shows a correlation between combined MS-275 and Doxorubicin treatment and downregulation of gene sets important for chromosomal regulation
    Figure Legend Snippet: HDAC inhibitors increase susceptibility of EwS to first line chemotherapeutics. a Heatmaps showing excess over Bliss synergy scores for combination treatment of Doxorubicin with HDACi FK228 (left) or MS-275 (right) in CHLA-10 or SK-N-MC cells. Scores greater than 1 (shown in red) denote synergistic combinations, whereas scores less than 1 (shown in green) denote antagonistic combinations. Data represent 3 technical triplicates. b Western blot analysis of caspase 3 and PARP as a measure of apoptosis susceptibility after treatment with Doxorubicin and HDACi, either in combination or individually. CHLA-10 were treated for 36 h and SK-N-MC for 24 h. Tubulin was used as loading control. c Focused heat map of genes, ≥1.5-fold differentially expressed in EwS line EW7 after combined treatment with DMSO, HDACi and/or first line chemotherapeutics Doxorubicin or Vincristine for 17 h. Treatment with Doxorubicin or Vincristine and MS-275, respectively, are highlighted in red. Each column represents one individual array. Microarray data with their normalized fluorescent signal intensities were used (robust multichip average (RMA); GSE162785). d GSEA enrichment plots of up- and downregulated gene sets after combined treatment with MS-275 and Doxorubicin. NES: normalized enrichment score. GSEA: http://www.broadinstitute.org/gsea/index.jsp . e GSEA leading-edge analysis of identified gene sets (C5_all, GO gene sets) downregulated after combined treatment with MS-275 and Doxorubicin. Set-to-set analysis shows a correlation between combined MS-275 and Doxorubicin treatment and downregulation of gene sets important for chromosomal regulation

    Techniques Used: Western Blot, Microarray

    28) Product Images from "Hepcidin is regulated by promoter-associated histone acetylation and HDAC3"

    Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00500-z

    Enrichment of HDAC3 at the hepcidin locus and effects of HDAC3 inhibition on genome-wide RNA expression profile. a Six-week-old C57Bl/6 male mice were administered Epo daily for 3 days 200 IU i.p. or control. ChIP-qPCR for HDAC3 (compared with IgG isotype control) at the hepcidin gene locus, along with the Cdkn1a locus as a positive control and a negative control genomic region. N = 3, paired t -tests. RNA sequencing of livers from mice treated with 3 weeks low-iron diet followed by either vehicle or two doses of RGFP966. Three biologic samples (those with the highest quality RNA) were selected for sequencing from each group. b Smear plot (log fold change vs expression levels), highlighting Hamp and Cdkn1a genes. c Volcano plot ( P -value vs log fold change) highlighting Hamp and Cdkn1a genes. d HDAC3 ChIP-Seq peaks were annotated and compared to differentially expressed genes. Expression of each HDAC in 6-week-old mice treated with e Epo vs control, and f ID vs control. Significance testing adjusted for multiple comparisons. N = 8 per group, same mice as experiments presented in Fig. 4
    Figure Legend Snippet: Enrichment of HDAC3 at the hepcidin locus and effects of HDAC3 inhibition on genome-wide RNA expression profile. a Six-week-old C57Bl/6 male mice were administered Epo daily for 3 days 200 IU i.p. or control. ChIP-qPCR for HDAC3 (compared with IgG isotype control) at the hepcidin gene locus, along with the Cdkn1a locus as a positive control and a negative control genomic region. N = 3, paired t -tests. RNA sequencing of livers from mice treated with 3 weeks low-iron diet followed by either vehicle or two doses of RGFP966. Three biologic samples (those with the highest quality RNA) were selected for sequencing from each group. b Smear plot (log fold change vs expression levels), highlighting Hamp and Cdkn1a genes. c Volcano plot ( P -value vs log fold change) highlighting Hamp and Cdkn1a genes. d HDAC3 ChIP-Seq peaks were annotated and compared to differentially expressed genes. Expression of each HDAC in 6-week-old mice treated with e Epo vs control, and f ID vs control. Significance testing adjusted for multiple comparisons. N = 8 per group, same mice as experiments presented in Fig. 4

    Techniques Used: Inhibition, Genome Wide, RNA Expression, Mouse Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, RNA Sequencing Assay, Sequencing, Expressing

    In vitro effects of PB on HAMP expression in liver-derived cells. a Effect of Panobinostat 10 and 100 nM on BMP9- (0, 10, and 50 ng/ml) induced Hamp1 expression (relative to Hprt ) in mouse precision cut liver slices. Data are normalized to baseline (untreated cells). N = 3 separate experiments. Two-way ANOVA by BMP9 dose and PB treatment. b Effect of Panobinostat 10 nM and BMP6 (0, 6, and 18 nM) on HAMP expression (relative to GAPDH ) in HuH7 human hepatoma cells. Data are normalized to baseline for each condition. N = 3 separate experiments. Two-way ANOVA by BMP6 dose and PB treatment. c Effects of PB treatment vs DMSO on HAMP , ID1 , SMAD7 , and ATOH8 mRNA expression in Huh7 cells, n = 3 separate experiments (same experiment as BMP6 0 nM condition from b . d Effect on HAMP and ID1 mRNA of treatment with isoform-specific HDAC inhibitors in Huh7 cells. HDAC(s) targeted by each inhibitor presented in the text. N = 3 separate experiments. One-way ANOVA, t -test for comparison between RGFP966 and control. e Whole-cell H3K9ac in Huh7 cells treated with RGFP966 and PB. f Depiction of HAMP promoter luciferase activity from knockdown of each HDAC isoform in a previously published RNAi screen (two replicates per HDAC). g Validation of HDAC3 and HDAC10 knockdown effects on HAMP mRNA expression in Huh7 cells ( n = 3 separate experiments, paired t -test). Data are means ± s.e.m. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; NS, P > 0.05
    Figure Legend Snippet: In vitro effects of PB on HAMP expression in liver-derived cells. a Effect of Panobinostat 10 and 100 nM on BMP9- (0, 10, and 50 ng/ml) induced Hamp1 expression (relative to Hprt ) in mouse precision cut liver slices. Data are normalized to baseline (untreated cells). N = 3 separate experiments. Two-way ANOVA by BMP9 dose and PB treatment. b Effect of Panobinostat 10 nM and BMP6 (0, 6, and 18 nM) on HAMP expression (relative to GAPDH ) in HuH7 human hepatoma cells. Data are normalized to baseline for each condition. N = 3 separate experiments. Two-way ANOVA by BMP6 dose and PB treatment. c Effects of PB treatment vs DMSO on HAMP , ID1 , SMAD7 , and ATOH8 mRNA expression in Huh7 cells, n = 3 separate experiments (same experiment as BMP6 0 nM condition from b . d Effect on HAMP and ID1 mRNA of treatment with isoform-specific HDAC inhibitors in Huh7 cells. HDAC(s) targeted by each inhibitor presented in the text. N = 3 separate experiments. One-way ANOVA, t -test for comparison between RGFP966 and control. e Whole-cell H3K9ac in Huh7 cells treated with RGFP966 and PB. f Depiction of HAMP promoter luciferase activity from knockdown of each HDAC isoform in a previously published RNAi screen (two replicates per HDAC). g Validation of HDAC3 and HDAC10 knockdown effects on HAMP mRNA expression in Huh7 cells ( n = 3 separate experiments, paired t -test). Data are means ± s.e.m. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; NS, P > 0.05

    Techniques Used: In Vitro, Expressing, Derivative Assay, Luciferase, Activity Assay

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    Selleck Chemicals lmk235
    Model for the role of HDAC5 in regulating CXCR4 expression in human HSC/HPCs. HDAC5 inhibition by <t>LMK235</t> results in enhanced acetylation of histone 3 on lysine 9 and histone 4 on lysine 16, as well as p65 acetylation. Acetylation of histone facilitates chromatin remodeling, whereas acetylation of p65 enhances its transcriptional activity. Acetylated p65 binds to CXCR4 promoter region and induces higher expression of CXCR4 and thus enhances HSC/HPC homing and engraftment
    Lmk235, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals class i hdac inhibitor ms 275
    <t>MS-275</t> suppresses pulmonary arterial remodeling, excessive extracellular matrix (ECM) accumulation and the increase of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) through restoring miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a Hematoxylin and eosin staining of pulmonary arteries (×400 magnification). b Quantitative morphometric analysis of the medial wall thickness of small pulmonary arteries. c Masson’s trichrome staining of pulmonary arteries (×400 magnification). d Changes of RVSP and RVHI in each group, RVHI = RV/(LV + S) (n = 6–8). * P
    Class I Hdac Inhibitor Ms 275, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals hdac inhibitors
    Neither <t>HDAC</t> nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM <t>vorinostat</t> for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).
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    Selleck Chemicals fk228
    Chromopeptide A induces apoptosis in prostate cancer cells. (A, B) Effects of chromopeptide A on apoptosis induction. PC3 and DU145 cells were treated with chromopeptide A or <t>FK228</t> at indicated concentrations for 48 h. Apoptosis was determined with the annexin-V/PI assay (A). Bars represent the mean±SD. * P
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    Model for the role of HDAC5 in regulating CXCR4 expression in human HSC/HPCs. HDAC5 inhibition by LMK235 results in enhanced acetylation of histone 3 on lysine 9 and histone 4 on lysine 16, as well as p65 acetylation. Acetylation of histone facilitates chromatin remodeling, whereas acetylation of p65 enhances its transcriptional activity. Acetylated p65 binds to CXCR4 promoter region and induces higher expression of CXCR4 and thus enhances HSC/HPC homing and engraftment

    Journal: Nature Communications

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    doi: 10.1038/s41467-018-05178-5

    Figure Lengend Snippet: Model for the role of HDAC5 in regulating CXCR4 expression in human HSC/HPCs. HDAC5 inhibition by LMK235 results in enhanced acetylation of histone 3 on lysine 9 and histone 4 on lysine 16, as well as p65 acetylation. Acetylation of histone facilitates chromatin remodeling, whereas acetylation of p65 enhances its transcriptional activity. Acetylated p65 binds to CXCR4 promoter region and induces higher expression of CXCR4 and thus enhances HSC/HPC homing and engraftment

    Article Snippet: For small molecule compound screen, 50,000 CD34+ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated.

    Techniques: Expressing, Inhibition, Activity Assay

    Inhibition of HDAC5 promotes CXCR4 transcription. a CXCR4 mRNA levels in M344- or LMK235-treated human CB CD34 + cells, relative to vehicle-treated cells, as assessed by quantitative RT-PCR. None indicates the group without any treatment. Data pooled from two independent experiments are shown ( n = 6, one-way ANOVA, *** p

    Journal: Nature Communications

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    doi: 10.1038/s41467-018-05178-5

    Figure Lengend Snippet: Inhibition of HDAC5 promotes CXCR4 transcription. a CXCR4 mRNA levels in M344- or LMK235-treated human CB CD34 + cells, relative to vehicle-treated cells, as assessed by quantitative RT-PCR. None indicates the group without any treatment. Data pooled from two independent experiments are shown ( n = 6, one-way ANOVA, *** p

    Article Snippet: For small molecule compound screen, 50,000 CD34+ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated.

    Techniques: Inhibition, Quantitative RT-PCR

    HDAC5 inhibition results in elevated levels of p65 acetylation. a Histogram of intracellular acetylated p65 (Ac-p65) levels of human CB CD34 + cells treated with vehicle, M344, or HDAC5 inhibitor LMK235. Representative histogram from three independent experiments is shown. b Mean fluorescence intensity (MFI) of intracellular acetylated p65 (Ac-p65) levels in vehicle, C646 (50 μM), M344-, M344 + C646-, LMK235-, LMK235 + C646-treated human CB CD34 + cells, as assessed by flow cytometry. Data pooled from two independent experiments are shown ( n = 4, one-way ANOVA, *** p

    Journal: Nature Communications

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    doi: 10.1038/s41467-018-05178-5

    Figure Lengend Snippet: HDAC5 inhibition results in elevated levels of p65 acetylation. a Histogram of intracellular acetylated p65 (Ac-p65) levels of human CB CD34 + cells treated with vehicle, M344, or HDAC5 inhibitor LMK235. Representative histogram from three independent experiments is shown. b Mean fluorescence intensity (MFI) of intracellular acetylated p65 (Ac-p65) levels in vehicle, C646 (50 μM), M344-, M344 + C646-, LMK235-, LMK235 + C646-treated human CB CD34 + cells, as assessed by flow cytometry. Data pooled from two independent experiments are shown ( n = 4, one-way ANOVA, *** p

    Article Snippet: For small molecule compound screen, 50,000 CD34+ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated.

    Techniques: Inhibition, Fluorescence, Flow Cytometry, Cytometry

    NF-κB signaling pathway is involved in HDAC5-mediated CB HSC homing. a Mean fluorescence intensity (MFI) of surface CXCR4 in vehicle, Andrographolide (Andro, 10 μM), M344-, M344 + Andro-, LMK235-, LMK235 + Andro-treated human CB CD34 + cells, as assessed by flow cytometry. Data pooled from three independent experiments are shown ( n = 6, one-way ANOVA, ** p

    Journal: Nature Communications

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    doi: 10.1038/s41467-018-05178-5

    Figure Lengend Snippet: NF-κB signaling pathway is involved in HDAC5-mediated CB HSC homing. a Mean fluorescence intensity (MFI) of surface CXCR4 in vehicle, Andrographolide (Andro, 10 μM), M344-, M344 + Andro-, LMK235-, LMK235 + Andro-treated human CB CD34 + cells, as assessed by flow cytometry. Data pooled from three independent experiments are shown ( n = 6, one-way ANOVA, ** p

    Article Snippet: For small molecule compound screen, 50,000 CD34+ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated.

    Techniques: Fluorescence, Flow Cytometry, Cytometry

    Inhibition of HDAC5 increases long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or LMK235 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or LMK235. The data were pooled from two independent experiments ( n = 10 mice per group, t -test, * p

    Journal: Nature Communications

    Article Title: Neutralizing negative epigenetic regulation by HDAC5 enhances human haematopoietic stem cell homing and engraftment

    doi: 10.1038/s41467-018-05178-5

    Figure Lengend Snippet: Inhibition of HDAC5 increases long-term engraftment of human CB HSC. a Representative flow cytometric analysis of human engraftment in the BM of NSG mice, 16 weeks after transplantation. Left is from a mouse without transplantation (negative control), center and right are from mice transplanted with human CB CD34 + cells treated with vehicle control or LMK235 for 16 h. Human engraftment was assessed as the percentage of human CD45 + cells. b The percentage of human CD45 + cells, B-cell (CD19 + ), and myeloid cell (CD33 + ) chimerism in the BM of NSG mice after transplantation with 10,000 CB CD34 + cells that had been treated with vehicle or LMK235. The data were pooled from two independent experiments ( n = 10 mice per group, t -test, * p

    Article Snippet: For small molecule compound screen, 50,000 CD34+ cells were cultured in the above medium with compounds (1 μM) from the SCREEN-WELL Epigenetics Library (Enzo Life Sciences, Farmingdale, NY, USA) for 16 h. For treatment of HDAC inhibitors, the cells were incubated with M344 (S2779, 1 μM) or LMK235 (S7569, 1 μM) (Selleck Chemicals, Houston, TX, USA) for 16 h unless otherwise stated.

    Techniques: Inhibition, Flow Cytometry, Mouse Assay, Transplantation Assay, Negative Control

    MS-275 suppresses pulmonary arterial remodeling, excessive extracellular matrix (ECM) accumulation and the increase of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) through restoring miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a Hematoxylin and eosin staining of pulmonary arteries (×400 magnification). b Quantitative morphometric analysis of the medial wall thickness of small pulmonary arteries. c Masson’s trichrome staining of pulmonary arteries (×400 magnification). d Changes of RVSP and RVHI in each group, RVHI = RV/(LV + S) (n = 6–8). * P

    Journal: Respiratory Research

    Article Title: Inhibition of HDAC1 alleviates monocrotaline-induced pulmonary arterial remodeling through up-regulation of miR-34a

    doi: 10.1186/s12931-021-01832-7

    Figure Lengend Snippet: MS-275 suppresses pulmonary arterial remodeling, excessive extracellular matrix (ECM) accumulation and the increase of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) through restoring miR-34a level in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats. a Hematoxylin and eosin staining of pulmonary arteries (×400 magnification). b Quantitative morphometric analysis of the medial wall thickness of small pulmonary arteries. c Masson’s trichrome staining of pulmonary arteries (×400 magnification). d Changes of RVSP and RVHI in each group, RVHI = RV/(LV + S) (n = 6–8). * P

    Article Snippet: Class I HDAC inhibitor MS-275 (Selleck Chemicals, USA) was dissolved in dimethyl sulfoxide (DMSO) and then diluted with normal saline to a final concentration of 6 mg/mL. miR-34a agomiR (GenePharm, Shanghai, China) was diluted with diethyl pyrocarbonate (DEPC) to a final concentration of 25 μM.

    Techniques: Staining

    Protocol for generation of rat pulmonary arterial hypertension (PAH) and reagent intervention models. The rat PAH model was induced by intraperitoneally injection of monocrotaline (MCT) (60 mg/kg) on day 1. MS-275 (3 mg/kg) was administrated to rats by intraperitoneally injection every other day for 28 days after MCT injection. miR-34a agomiR (25 μM) was given to rats via tail vein injection on days 1, 7, 14, 21 and 28 respectively after MCT injection

    Journal: Respiratory Research

    Article Title: Inhibition of HDAC1 alleviates monocrotaline-induced pulmonary arterial remodeling through up-regulation of miR-34a

    doi: 10.1186/s12931-021-01832-7

    Figure Lengend Snippet: Protocol for generation of rat pulmonary arterial hypertension (PAH) and reagent intervention models. The rat PAH model was induced by intraperitoneally injection of monocrotaline (MCT) (60 mg/kg) on day 1. MS-275 (3 mg/kg) was administrated to rats by intraperitoneally injection every other day for 28 days after MCT injection. miR-34a agomiR (25 μM) was given to rats via tail vein injection on days 1, 7, 14, 21 and 28 respectively after MCT injection

    Article Snippet: Class I HDAC inhibitor MS-275 (Selleck Chemicals, USA) was dissolved in dimethyl sulfoxide (DMSO) and then diluted with normal saline to a final concentration of 6 mg/mL. miR-34a agomiR (GenePharm, Shanghai, China) was diluted with diethyl pyrocarbonate (DEPC) to a final concentration of 25 μM.

    Techniques: Injection

    MS-275 alleviates monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. a Right ventricular systolic pressure (RVSP) waveform. b Changes of RVSP in each group (n = 6–8). c Changes of right ventricular hypertrophy index (RVHI) in each group, RVHI = RV/(LV + S) (n = 6–8). * P

    Journal: Respiratory Research

    Article Title: Inhibition of HDAC1 alleviates monocrotaline-induced pulmonary arterial remodeling through up-regulation of miR-34a

    doi: 10.1186/s12931-021-01832-7

    Figure Lengend Snippet: MS-275 alleviates monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats. a Right ventricular systolic pressure (RVSP) waveform. b Changes of RVSP in each group (n = 6–8). c Changes of right ventricular hypertrophy index (RVHI) in each group, RVHI = RV/(LV + S) (n = 6–8). * P

    Article Snippet: Class I HDAC inhibitor MS-275 (Selleck Chemicals, USA) was dissolved in dimethyl sulfoxide (DMSO) and then diluted with normal saline to a final concentration of 6 mg/mL. miR-34a agomiR (GenePharm, Shanghai, China) was diluted with diethyl pyrocarbonate (DEPC) to a final concentration of 25 μM.

    Techniques:

    Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).

    Journal: Oncotarget

    Article Title: Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

    doi:

    Figure Lengend Snippet: Neither HDAC nor BAP1 depletion alters total cellular HDAC activity HDAC activity was assessed in A549 cells A–C. and MSTO-211H cells D–F. As positive controls, cells were A. chronically treated with 250 nM of the pan-HDAC inhibitor TSA for 16 hr (mean of two independent experiments), or D. acutely treated with 5 μM vorinostat for 2 hr (mean of three independent experiments, error bar SD). BAP1 depletion does not affect activity towards the HDAC-GloI/II substrate. Cells were reverse transfected with 20 nM siRNA as indicated for 48 hr. Immunoblotting shows efficient depletion by reverse transfection of siRNA in the 96-well assay format, B. E., the percentage efficiency for depletion of the target protein (KD%) and the ratio of HDAC2/HDAC1 expression (H2/H1) is indicated. For the HDAC-Glo assay run in parallel, C. F., data show the mean of three C. or four F. independent experiments (error bars SD, no significant difference was determined between samples using one-way ANOVA with Dunnett's post-hoc test).

    Article Snippet: Cells were treated with vehicle (0.1% DMSO) or HDAC inhibitors at their LC30 (2.5 μM vorinostat, 1.25 μM mocetinostat, 20 μM MC1568) for the final 48 hr prior to analysis, with the media and drug replaced every 24 hr.

    Techniques: Activity Assay, Transfection, Expressing, Glo Assay

    BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).

    Journal: Oncotarget

    Article Title: Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

    doi:

    Figure Lengend Snippet: BAP1 status alters sensitivity of mesothelioma cells to HDAC inhibitors A. Dose response curves for HDAC inhibitors in MSTO-211H cells. Vorinostat (broad spectrum), mocetinostat (class I) and MC1568 (Class IIa) were added to cells at the indicated concentrations for 48 hr prior to CellTitre-Glo assay (error bars show SD of 4 technical replicates). MC1568 remains in aqueous solution at concentrations below 50 μM. B–D. HDAC2 or BAP1 depletion sensitizes cells to HDAC inhibitors. MSTO-211H cells were reverse transfected with 20 nM siRNA for 72 hr, and treated with vehicle (0.1% DMSO) B. or with HDAC inhibitors C. D. for the final 48 hr, before viable cell estimation by CellTitre-Glo assay (error bars show SD for three independent experiments). B. Treatment with vehicle: relative viability is plotted, compared to cells transfected with the siC control and treated with vehicle (one-way ANOVA with Tukey's post-hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). C. Treatment with the indicated HDAC inhibitors at their LC 30 : 20 μM MC1568, 2.5 μM vorinostat and 1.25 μM mocetinostat. The relative viability is plotted as a histogram for each drug treatment, this is based on comparison to cells transfected with siC and treated with that drug. For each bar on the histograms, percentage viability is also indicated, which compares the drug treated cells to the DMSO treated cells (shown in B) for the same knockdown condition; statistical significance for this comparison is shown (one-sample t -test, * P ≤ 0.05, ** P ≤ 0.01). D. Dose response curves for vorinostat in HDAC2 and/or BAP1 depleted MSTO-211H cells; all data are normalized to siC transfected cells in 0.1% DMSO. E. Dose response curves for mocetinostat in a panel of mesothelioma cell lines with high (MSTO-211H) or low (other cell lines) constitutive HDAC2 expression. Data are normalized to 0.1 μM treatment for each cell line (error bars show SD for three independent experiments).

    Article Snippet: Cells were treated with vehicle (0.1% DMSO) or HDAC inhibitors at their LC30 (2.5 μM vorinostat, 1.25 μM mocetinostat, 20 μM MC1568) for the final 48 hr prior to analysis, with the media and drug replaced every 24 hr.

    Techniques: Glo Assay, Transfection, Expressing

    Chromopeptide A induces apoptosis in prostate cancer cells. (A, B) Effects of chromopeptide A on apoptosis induction. PC3 and DU145 cells were treated with chromopeptide A or FK228 at indicated concentrations for 48 h. Apoptosis was determined with the annexin-V/PI assay (A). Bars represent the mean±SD. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A induces apoptosis in prostate cancer cells. (A, B) Effects of chromopeptide A on apoptosis induction. PC3 and DU145 cells were treated with chromopeptide A or FK228 at indicated concentrations for 48 h. Apoptosis was determined with the annexin-V/PI assay (A). Bars represent the mean±SD. * P

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques:

    Chromopeptide A impairs prostate cancer cell migration in a dose-dependent manner. (A, B) PC3 cells were planted in the upper transwell chamber and treated with indicated concentrations of chromopeptide A or FK228. Cells were allowed to migrate for 24 h; then, migrated cells were stained and observed under a microscope (×200) (A) and cell numbers were counted (B). Bars represent the mean±SD. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A impairs prostate cancer cell migration in a dose-dependent manner. (A, B) PC3 cells were planted in the upper transwell chamber and treated with indicated concentrations of chromopeptide A or FK228. Cells were allowed to migrate for 24 h; then, migrated cells were stained and observed under a microscope (×200) (A) and cell numbers were counted (B). Bars represent the mean±SD. * P

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques: Migration, Staining, Microscopy

    Chromopeptide A impacts the viability of prostate cancer cells through targeting HDAC. (A) Effects of chromopeptide A and FK228 on cell proliferation. Prostate cancer cell lines PC3, DU145 and LNCaP were treated with chromopeptide A or FK228 at the indicated concentrations for 72 h. Cell viability was assessed with the SRB assay. Bars represent the mean±SD. (B, C) Effects of chromopeptide A and FK228 on HDAC activity inhibition. PC3 and DU145 were treated with chromopeptide A or FK228 (0.2, 1, 5, 20, and 50 nmol/L) for 24 h. HDAC activity was measured (B) and the substrate of class I HDAC was detected with immunoblotting (C). Bars represent the means±SD. ** P

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A impacts the viability of prostate cancer cells through targeting HDAC. (A) Effects of chromopeptide A and FK228 on cell proliferation. Prostate cancer cell lines PC3, DU145 and LNCaP were treated with chromopeptide A or FK228 at the indicated concentrations for 72 h. Cell viability was assessed with the SRB assay. Bars represent the mean±SD. (B, C) Effects of chromopeptide A and FK228 on HDAC activity inhibition. PC3 and DU145 were treated with chromopeptide A or FK228 (0.2, 1, 5, 20, and 50 nmol/L) for 24 h. HDAC activity was measured (B) and the substrate of class I HDAC was detected with immunoblotting (C). Bars represent the means±SD. ** P

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques: Sulforhodamine B Assay, Activity Assay, Inhibition

    Chromopeptide A induces G 2 /M cell cycle arrest in prostate cancer cells. (A) Effects of chromopeptide A and FK228 on cell cycle distribution. PC3 and LNCaP cells were treated with chromopeptide A or FK228 at the indicated concentrations for 24 h. The cell cycle distribution was analyzed by FACS after propidium iodide staining. Quantification results are presented. Bars represent the mean±SD. (B) Impacts of chromopeptide A and FK228 on G 2 /M transition regulators. PC3 and LNCaP cells were treated with chromopeptide A or FK228 at indicated concentrations for 24 h, and cells lysates were immunoblotted with the indicated antibodies.

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A induces G 2 /M cell cycle arrest in prostate cancer cells. (A) Effects of chromopeptide A and FK228 on cell cycle distribution. PC3 and LNCaP cells were treated with chromopeptide A or FK228 at the indicated concentrations for 24 h. The cell cycle distribution was analyzed by FACS after propidium iodide staining. Quantification results are presented. Bars represent the mean±SD. (B) Impacts of chromopeptide A and FK228 on G 2 /M transition regulators. PC3 and LNCaP cells were treated with chromopeptide A or FK228 at indicated concentrations for 24 h, and cells lysates were immunoblotted with the indicated antibodies.

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques: FACS, Staining

    Chromopeptide A is a novel class I HDAC inhibitor. (A) Chemical structures of chromopeptide A and FK228. (B) Selectivity profiling on HDAC enzymes of chromopeptide A and FK228. The indicated concentrations of chromopeptide A and FK228 were exposed to HDAC1-8 and the enzyme activity was determined by the protease-coupled assay. Bars represent the mean±SD. (C) Kinetic analysis of chromopeptide A and FK228 on HDAC1. Chromopeptide A and FK228 were exposed to different concentrations of Ac-peptide-AMC substrates with HDAC1. The alpha values and K i values were determined in a mixed competitive inhibition fitted model. Bars represent the mean±SD.

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A is a novel class I HDAC inhibitor. (A) Chemical structures of chromopeptide A and FK228. (B) Selectivity profiling on HDAC enzymes of chromopeptide A and FK228. The indicated concentrations of chromopeptide A and FK228 were exposed to HDAC1-8 and the enzyme activity was determined by the protease-coupled assay. Bars represent the mean±SD. (C) Kinetic analysis of chromopeptide A and FK228 on HDAC1. Chromopeptide A and FK228 were exposed to different concentrations of Ac-peptide-AMC substrates with HDAC1. The alpha values and K i values were determined in a mixed competitive inhibition fitted model. Bars represent the mean±SD.

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques: Activity Assay, Inhibition

    Chromopeptide A inhibits PC3 tumor growth in vivo . (A) Tumor growth inhibition in chromopeptide A-treated PC3 xenografts. Mice bearing PC3 cells were treated with chromopeptide A or FK228 (iv, once a week) for 18 d after the tumor volume reached 100–150 mm 3 . Tumor volumes were measured every three days and are presented as the average relative tumor volume±SD. The percentage of tumor volume inhibition was measured compared with the vehicle group. Bars represent the mean±SD. * P

    Journal: Acta Pharmacologica Sinica

    Article Title: Marine-derived chromopeptide A, a novel class I HDAC inhibitor, suppresses human prostate cancer cell proliferation and migration

    doi: 10.1038/aps.2016.139

    Figure Lengend Snippet: Chromopeptide A inhibits PC3 tumor growth in vivo . (A) Tumor growth inhibition in chromopeptide A-treated PC3 xenografts. Mice bearing PC3 cells were treated with chromopeptide A or FK228 (iv, once a week) for 18 d after the tumor volume reached 100–150 mm 3 . Tumor volumes were measured every three days and are presented as the average relative tumor volume±SD. The percentage of tumor volume inhibition was measured compared with the vehicle group. Bars represent the mean±SD. * P

    Article Snippet: FK228 was obtained from Selleck Chemicals (Shanghai, China).

    Techniques: In Vivo, Inhibition, Mouse Assay