Journal: PLOS ONE

Article Title: Identification of therapeutic sensitivities in a spheroid drug combination screen of Neurofibromatosis Type I associated High Grade Gliomas

doi: 10.1371/journal.pone.0277305

Figure Lengend Snippet: (A) Representative images of glioma line 5746 treated with GDC-0941 (1000nM), Vorinostat (500nM) or a combination. Images on the left show size and red intensity of the sphere at the starting point of the assay (0h). Images on the right show the same sphere at 72h. Combination of GDC-0941 and Vorinostat causes potent cell death. (B) Growth curves of 5746 treated with GDC-0941 and/or Vorinostat for 72h. Error bars denote the standard deviation. A log2(fold change) of -1 denotes 50% lower signal compared to starting point. (C) Western blot confirming the target inhibition of our compounds. An increase in ubiquitinated proteins is shown by treatment of cell line 17 with 5nM bortezomib; pCHEK1 is inhibited with 100nM LY2606368; MYC is inhibited by the BRD4 inhibitor JQ1 (500nM); BMI1 is downregulated by treatment with 500nM PTC596; H3K27Ac is upregulated by treatment with 1μM Vorinostat and expression of CDK9 and its substrate pS2 RNApol II is reduced with 25nM Dinaciclib. (D) 3D graphical representation of the synergy score as determined by synergy finder. X-axis: GDC-0941 concentration, Y-axis Vorinostat concentration, Z-axis synergy score (δ). (E) 2D heatmap representing the same synergy score data (D).

Article Snippet: The following antibodies were purchased from Cell signaling Technologies: pERK (Catalogue # 4370), total ERK (Catalogue # 9102), pAKT (Catalogue # 4060), total AKT (Catalogue # 4691), Ubiquitin (Catalogue # 4289), MYC (Catalogue # 18583), pCHEK1 S296 (Catalogue # 90178), BMI1 (Catalogue # 5856), H3K27Ac (Catalogue # 8173), total H3 (Catalogue # 14269), CDK9 (Catalogue # 2316), Vinculin (Catalogue # 4650), CPNase (Catalogue # 5664) and MAG (Catalogue # 9043).

Techniques: Standard Deviation, Western Blot, Inhibition, Expressing, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Selectivity of Hydroxamate- and Difluoromethyloxadiazole-Based Inhibitors of Histone Deacetylase 6 In Vitro and in Cells

doi: 10.3390/ijms24054720

Figure Lengend Snippet: Tubulin/histone deacetylation inhibition assay. Panel ( A ) RPMI-8226 cells were treated with dilution series of HDAC6is for six hours and levels of Ac-tubulin and Ac-H3 were determined by Western blotting (ACY-1215 shown as a representative). Panel ( B ) Acetylation values were normalized to total tubulin and plotted as relative tubulin acetylation. IC 50 values were calculated from fitted curves. The potency of tested compounds to inhibit tubulin deacetylation ranges from 136 nM to 1.7 μM for TSA and Tubacin, respectively. Panel ( C ) Quantification of the effect of inhibitors on the acetylation status of histone H3. Panel ( D ) Comparison of EC 50 values for tubulin deacetylation and nanoBRET HDAC6 assays.

Article Snippet: Antibodies used were anti-alpha tubulin (1:4000 dilution, rabbit, #Ab18251, Abcam, Cambridge, UK), anti-acetylated tubulin (1:2500 dilution, mouse, #T7451, Sigma-Aldrich, St. Louis, MO, USA), and anti-acetylated histone H3 (Lys9/Lys14) (1:2000 dilution, rabbit, #9677, Cell Signaling, Danvers, MA, USA).

Techniques: Inhibition, Western Blot

Journal: Cancers

Article Title: SMAC Mimetics Synergistically Cooperate with HDAC Inhibitors Enhancing TNF-α Autocrine Signaling

doi: 10.3390/cancers15041315

Figure Lengend Snippet: Tolinapant inhibits cIAP1 and XIAP, and entinostat acetylate Histone H3. ( A , B ) Changes in cIAP1 and acetylated histone H3 were measured with Western blot in OVCAR8 after 24 h treatment with either single-agent or combination tolinapant 25 μM and entinostat 2 μM. cIAP1 was degradated by tolinapant. Histone H3 was acetylated with entinostat treatment. ( C ) Changes in protein–protein interaction between SMAC and XIAP Western blot after co-immune precipitation using anti XIAP antibody in OVCAR3 and OVCAR8 after 24 h treatment with either single-agent or combination tolinapant 25 μM and entinostat 2 μM. Tolinapant did not change the protein level of XIAP and SMAC, but conjugation between SMAC and XIAP was completely inhibited.

Article Snippet: Antibodies for RelA (NF-kB p65 #8242S CST), p50 (NF-kBp105/50 #13586S), RelB (#10544 CST), p52 (NF-kB p100/52 #37359S CST), Acetyl-Histone H3 (K27, #4353S CST), and SP3 (sc-365220 Santa Cruz).

Techniques: Western Blot, Conjugation Assay

Journal: Cancers

Article Title: SMAC Mimetics Synergistically Cooperate with HDAC Inhibitors Enhancing TNF-α Autocrine Signaling

doi: 10.3390/cancers15041315

Figure Lengend Snippet: Entinostat enhances the production of TNF-α through acetylation of TNF-α promoter region. ( A ) There are three NFkB binding sites and 6 SP3 binding sites on the TNF-α promoter region. ( B ) Chromatin-IP (ChIP) qPCR assay using anti-acetylated histone H3 (AcH3) antibody. All isotype controls (IgG) were under 0.01% of input. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM) 18 h treatment. (* p < 0.05). ( C ) ChIP qPCR assay using anti-NFkB (RelA, p50, RelB, p52) antibodies. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM) 18 h treatment. ( D ) ChIP qPCR assay using anti-SP3 antibody. DNA samples we corrected from OVCAR8 cells after tolinapant (25 μM) alone or in combination with entinostat (2 μM), 18 h treatment. ( E ) Genomic locations of transcription factor binding sites in the promoter region for the TNF-α gene.

Article Snippet: Antibodies for RelA (NF-kB p65 #8242S CST), p50 (NF-kBp105/50 #13586S), RelB (#10544 CST), p52 (NF-kB p100/52 #37359S CST), Acetyl-Histone H3 (K27, #4353S CST), and SP3 (sc-365220 Santa Cruz).

Techniques: Binding Assay, Chromatin Immunoprecipitation

Journal: Journal of Cardiovascular Pharmacology

Article Title: HDAC Inhibitors Alleviate Uric Acid–Induced Vascular Endothelial Cell Injury by Way of the HDAC6/FGF21/PI3K/AKT Pathway

doi: 10.1097/FJC.0000000000001372

Figure Lengend Snippet: TSA attenuated UA-stimulated endothelial dysfunction in HUVECs. A, Representative cell morphology and ROS generation captured by a microscope (n = 3). B, TNF-α, IL-1β, and IL-6 mRNA levels determined by RT-qPCR (n = 5). C, NO production (n = 5). D, RT-qPCR analysis of CD31 and α-SMA (n = 5). E, WB analysis of CD31 and α-SMA (n = 4). F, α-SMA expression analyzed by immunofluorescence (n = 3). G, WB analysis of p-AKT/AKT and p-eNOS/eNOS (n = 4). H, RT-qPCR analysis of FGF21 (n = 5). I, WB analysis of FGF21 (n = 4). J, Level of histone H3 acetylation on the FGF21 promoter region, as analyzed by ChIP (n = 4). One-way ANOVA followed by Tukey's post hoc multiple-group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001 versus CON; # P < 0.05, ## P < 0.01, ### P < 0.001 versus 12 mg/dL UA; & P < 0.05, && P < 0.01 versus 12 mg/dL UA+TSA low.

Article Snippet: The remaining part was added with 100 μL ChIP buffer, reacted with acetylated histone H3 antibody (1:100, CST, #8173) or IgG (1:100, Santa Cruz, sc2025), and then mixed with the prepared magnetic beads for an incubation at 4°C overnight.

Techniques: Microscopy, Quantitative RT-PCR, Expressing, Immunofluorescence

Journal: Journal of Cardiovascular Pharmacology

Article Title: HDAC Inhibitors Alleviate Uric Acid–Induced Vascular Endothelial Cell Injury by Way of the HDAC6/FGF21/PI3K/AKT Pathway

doi: 10.1097/FJC.0000000000001372

Figure Lengend Snippet: Different from TSA, Mocetinostat, a class-Ⅰ selective HDAC inhibitor did not attenuate UA-stimulated endothelial dysfunction in HUVECs. A, HDAC1,2,3,6 activity detected by HDAC kits (n = 5). B, TNF-α, IL-1β, and IL-6 mRNA level detected by RT-qPCR (n = 5). C, NO production (n = 5). D, RT-qPCR analysis of FGF21 (n = 5). E, WB analysis of FGF21 (n = 4). F, Level of histone H3 acetylation on the FGF21 promoter region, as analyzed by ChIP (n = 4). One-way ANOVA followed by Tukey's post hoc multiple-group comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001 versus CON; # P < 0.05, ## P < 0.01, ### P < 0.001 versus 12 mg/dL UA; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 versus 12 mg/dL UA+TSA high.

Article Snippet: The remaining part was added with 100 μL ChIP buffer, reacted with acetylated histone H3 antibody (1:100, CST, #8173) or IgG (1:100, Santa Cruz, sc2025), and then mixed with the prepared magnetic beads for an incubation at 4°C overnight.

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Communications Biology

Article Title: The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity

doi: 10.1038/s42003-023-04485-y

Figure Lengend Snippet: a Quantitative reverse transcription PCR (qRT-PCR) of MHC class I and class II genes in BEAS2B cells treated for 3 days with 1, 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA; The acetylation mark (H3AcK14) was detected by immunoblotting. b Quantitative reverse transcription PCR (qRT-PCR) of MHC class I and class II genes in NBE1 cells treated for 3 days with 10, 100, 1000, 2000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA. The acetylation mark (H3AcK14) was detected by immunoblotting. c Flow cytometry analysis of extracellular HLA class I proteins in BEAS2B cells treated for 2 days with 1 µM zabadinostat or DMSO control, n = 4, results presented as mean values +/−SD, Student’s t test; The acetylation mark (H3AcK9) was detected by immunoblotting in BEAS2B cells. d Flow cytometry analysis of extracellular HLA class I proteins in NBE1 cells treated for 2 days with 1 µM zabadinostat or DMSO control, n = 4, results presented as mean values +/−SD, Student’s t test; The acetylation mark (H3AcK9) was detected by immunoblotting in NBE1 cells; Quantitative reverse transcription PCR (qRT-PCR) of MHC class I and class II genes in lung RNA from Balb/c mice treated for 14 days ( e ) and 30 days ( f ) with 10 mg/kg zabadinostat or DMSO control; n = 4; results presented as mean values +/−SD; one-way ANOVA. The acetylation mark (H3AcK9) of representative mouse samples was detected by immunoblotting.

Article Snippet: The following antibodies were used in immunoblotting: anti-Histone H3 (ab1791, Abcam; 1:1000), anti-H3AcK9 (ab10812, Abcam; 1:1000), anti-H3AcK14 (#7627, Cell Signaling, Danvers, MA, USA; 1:1000), anti-β-Actin (#3700, Cell Signaling; 1:1000), anti-CIITA (TA319682, Origene, Rockville, MD, USA; 1:1000) all overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Communications Biology

Article Title: The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity

doi: 10.1038/s42003-023-04485-y

Figure Lengend Snippet: a Quantitative reverse transcription PCR (qRT-PCR) of CIITA in BEAS2B cells treated for 3 days with 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA; The CIITA protein level and acetylation mark (H3AcK14) was detected by immunoblotting. b Quantitative reverse transcription PCR (qRT-PCR) of CIITA in NBE1 cells treated for 3 days with 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA; The CIITA protein level and acetylation mark (H3AcK14) was detected by immunoblotting. c Quantitative reverse transcription PCR (qRT-PCR) of MHC genes in BEAS2B cells treated for 2 days with 50 nM siCIITA and for 3 days with 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA. d Quantitative reverse transcription PCR (qRT-PCR) of MHC class I genes in NBE1 cells treated for 2 days with 50 nM siCIITA and for 3 days with 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA. e Quantitative reverse transcription PCR (qRT-PCR) of CIITA in BEAS2B cells treated for 2 days with 50 nM siCIITA and for 3 days with 10, 100, 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA. The CIITA protein level and acetylation mark (H3AcK14) were detected by immunoblotting and quantified. f Histone H3 and H3AcK9 ChIP on MHC gene promoters in BEAS2B cells treated for 2 days with 50 nM siCIITA and for 3 days with 1000 nM zabadinostat or DMSO control; n = 3; results presented as mean values +/−SD; one-way ANOVA.

Article Snippet: The following antibodies were used in immunoblotting: anti-Histone H3 (ab1791, Abcam; 1:1000), anti-H3AcK9 (ab10812, Abcam; 1:1000), anti-H3AcK14 (#7627, Cell Signaling, Danvers, MA, USA; 1:1000), anti-β-Actin (#3700, Cell Signaling; 1:1000), anti-CIITA (TA319682, Origene, Rockville, MD, USA; 1:1000) all overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot

Journal: Communications Biology

Article Title: The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity

doi: 10.1038/s42003-023-04485-y

Figure Lengend Snippet: a Flow cytometry analysis of the extracellular MHC class II protein level in bone marrow (collected from C57BL/6)-derived dendritic cells (CD11c+/CD86+) treated with 1 µM zabadinostat or DMSO control for 48 h; n = 5, Student’s t test; the acetylation mark (H3AcK14) and total histone 3 level were detected by flow cytometry; n = 5, Student’s t test. b Quantitative reverse transcription PCR (qRT-PCR) of MHC class I and II genes was also performed; n = 5; results presented as mean values +/−SD; one-way ANOVA. c Flow cytometry analysis of the extracellular MHC class II protein level in bone marrow (collected from Balb/c)-derived dendritic cells (CD11c+/CD86+) treated with 1 µM zabadinostat or DMSO control for 48 h, n = 5; the acetylation mark (H3AcK14) and total histone 3 level were detected by flow cytometry; n = 5; Student’s t test. d Activation of CD4, CD8 T cells and B cells upon 1 µM zabadinostat treatment or DMSO control was evaluated in splenocytes collected from Balb/c mice; n = 5; one-way ANOVA. e Viability of splenocytes measured by flow cytometry with L/D staining; graph represents pooled results from two animal experiments (see Fig. ( n = 4) and Supplementary Fig. ( n = 4)); results presented as mean values +/−SD.

Article Snippet: The following antibodies were used in immunoblotting: anti-Histone H3 (ab1791, Abcam; 1:1000), anti-H3AcK9 (ab10812, Abcam; 1:1000), anti-H3AcK14 (#7627, Cell Signaling, Danvers, MA, USA; 1:1000), anti-β-Actin (#3700, Cell Signaling; 1:1000), anti-CIITA (TA319682, Origene, Rockville, MD, USA; 1:1000) all overnight at 4 °C.

Techniques: Flow Cytometry, Derivative Assay, Quantitative RT-PCR, Activation Assay, Staining