Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 3. HDAC3 is required for extracellular matrix homeostasis and remodeling of semilunar valves. A, Movat’s pentachrome staining of remodeling aortic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO aortic valves with a reduction and remodeling of proteoglycans in control valves. B, Movat’s pentachrome staining of remodeling pulmonic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO pulmonic valves with a reduction and remodeling of proteoglycans in control valves. C, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 aortic valve. D, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 pulmonic valve. E, Verhoeff-Van Gieson (VVG) staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. F, Verhoeff-Van Gieson staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. G, Movat’s pentachrome-stained sections showincreasedproteoglycans(blue)inaorticvalvecuspsofE18.5Hdac3Mef2CKOhearts.H,Movat’spentachromestainingrevealsincreasedproteoglycans(blue) in E18.5 Hdac3Mef2CKO pulmonic valve. I, representative images of cleaved caspase-3 immunostaining in E13.5 semilunar valves. Arrows, positive staining. J, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO aortic valves. K, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO pulmonic valves. L, schematic model depicting disorganized extracellular matrix and reduced apoptosis in Hdac3Isl1KO E13.5 semilunar valves. M, schematic model showing hyperplastic, enlarged, and disorganized Hdac3Isl1KO E18.5 semilunar valves.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Staining, Control, Expressing, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 4. HDAC3 is a critical regulator of TGF- signaling pathway. A–E, IPA of microarray data from E9.5 Hdac3Isl1KO hearts. A, cardiovascular development and function subcategories significantly dysregulated in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. B, classes of congenital heart anomalies affected in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. C, 30 most significant upstream regulatorsfromIPAupstreamanalysisofE9.5Hdac3Isl1KOmicroarraydata.D,clusteredheatmapofdifferentiallyexpressedgenesrelatedtoupstreamregulator TGF-1 from IPA upstream analysis of Hdac3Isl1KO microarray data. E, top 30 upstream regulators, based on number of associated genes differentially expressed in E9.5 Hdac3Isl1KO hearts from IPA upstream analysis. F, transcripts for Sumo1, Nrp2, Smad4, Snai1, Tgf-1, and Kpnb1 were detected by real-time qPCR in Hdac3F/F and Hdac3Isl1KO outflow tract with right ventricle derived from E9.5 embryos (mean S.E. (error bars), n 3). G–J, ChIP-qPCR analysis of HDAC3 recruitment to promoter-proximal regions of TGF- pathway genes performed in wild-type E9.5 outflow tract (mean S.E., n 3). K and L, ELISA for TGF-1 (K) and phospho-SMAD2/3 (L) was performed in Hdac3F/F and Hdac3Isl1KO outflow tracts (mean S.E., n 3 (K) and n 4 (L)). M, phospho-SMAD2/3 immunostaining (arrows) in E9.5 Hdac3Isl1KO; R26R-LacZ/ hearts.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Microarray, Derivative Assay, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 8. HDAC3 epigenetically silences TGF-1 within valvular mesenchymal cells by recruiting PRC2 complex to the NCOR complex. A, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells (EC) or cardiac mesenchymal cells (MC) from E10.5 Hdac3F/F outflow tract cushion explants, infected either with CRE or GFP control lentivirus. B, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves (SL) derived from Hdac3Isl1KO and Hdac3F/F E14.5 hearts. C, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or cardiac mesen- chymal cells from E10.5 outflow tract cushion explants. D, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves from E14.5 hearts. E–K, ChIP-qPCR analysis of H3K27 trimethylation (E), H3K27 acetylation (F), RNA polymerase II (G), CREBBP (H), EZH2 (I), EED (J), and SUZ12 (K) upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. L, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA (sc-shRNA), EZH2 shRNA, or NCOR1 shRNA. M, ChIP-qPCR analysis of NCOR1 upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. N, Co-ChIP for HDAC3 and either EZH2, NCOR1, H3K27me3, H3K27ac, or polymerase II upstream of TGF-1 in E14.5 wild-type dissected semilunar valves. O, total lysates from E14.5 wild-type pooled semilunar valves were immunoprecipitated (IP) by EZH2 antibody, and Western blot was performed using HDAC3 antibody. -Tubulin is shown as an input control. HDAC3 was quantified and normalized to total input -tubulin using ImageJ software (mean S.E. (error bars), n 3). P, ChIP-qPCR analysis of H3K27me3 upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA, EZH2 shRNA, or NCOR1 shRNA.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Isolation, Infection, Control, Derivative Assay, ChIP-qPCR, shRNA, Immunoprecipitation, Western Blot, Software

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 9. HDAC3 functions in a deacetylase-independent manner to regulate EndMT and epigenetic silencing of TGF-1. A, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression constructs were transfected in HEK-293T cells. Expression was detected by Western blot from whole cell lysates using FLAG antibody. GAPDH is shown as a loading control. B, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified and normalized to total input GAPDH using ImageJ software (mean S.D. (error bars), n 3). C, HDAC activity of HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified against a pseudosubstrate. D, EndMT assay of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, imaged 24 h after isolation. E, quantification of average radial migration, measured in eight directions, of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, measured 24 h after isolation(mean S.E.(errorbars),n3).F,relativemRNAlevelsofTgf-1incontrol-orCre-infectedE14.5Hdac3F/Fvalvularmesenchymalcellsco-infectedwith control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3). G, a 1309-bp TGF-1 promoter luciferase reporter (WT) or a truncated, 1267-bp TGF-1 promoter luciferase reporter, lacking an HDAC3-enriched region (mutant) were transfected in murine endothelial cells with and without an HDAC3-FLAG or HDAC3H134A,H135A-FLAG expression plasmid. Induction is represented as a ratio of firefly and Renilla luciferase activity. H–L, ChIP-qPCR analysis of H3K27 acetylation (H), H3K27 trimethylation (I), EZH2 (J), EED (K), and SUZ12 (L) upstream of TGF-1 in control- or Cre-infected E14.5 Hdac3F/F valvular mesenchymal cells co-infected with control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3).

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Histone Deacetylase Assay, Expressing, Construct, Transfection, Western Blot, Control, Software, Activity Assay, Infection, Isolation, Migration, Luciferase, Mutagenesis, Plasmid Preparation, ChIP-qPCR

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 10. Summary of phenotypes and proposed model of HDAC3 function within second heart field progenitor cells and second heart field-derived mesenchymal cells. A, loss of HDAC3 in second heart field progenitor cells leads to outflow tract and semilunar valve pathologies. Strikingly, genetic deletion of HDAC3 in differentiated mesenchymal and smooth muscle cells (Hdac3TaglnKO) recapitulates the majority of these phenotypes. However, deletion of HDAC3 in differentiated cardiomyocytes (Hdac3Myh6KO) or endothelial cells (Hdac3Cdh5KO) did not recapitulate the cardiovascular defects observed in Hdac3Isl1KO embryos. B, Hdac3Isl1KO hearts exhibit disorganized collagen and elastin within dilated aortic walls and hyperplastic semilunar valves containing activated myofibroblasts and disorganized extracellular matrix. In both control and Hdac3Isl1KO cardiac endothelial cells, the upstream regulatory region of TGF-1 is occupied by RNA polymerase II and CREBBP and exhibits H3K27 acetylation concomitant with TGF-1 expression. In control semilunar valves, endothelial cells undergo EndMT to become mesen- chymalcells.Inthesemesenchymalcells,NCOR1,HDAC3,andPRC2complex(EZH2,EED,andSUZ12)arerecruitedtotheupstreamregulatoryregionofTGF-1,which becomes trimethylated on histone H3 Lys-27, and TGF-1 expression is epigenetically silenced. In Hdac3Isl1KO hearts, EZH2, EED, and SUZ12 are not recruited to the TGF-1 regulatory region, RNA polymerase II and CREBBP are present, and histone H3 Lys-27 remains acetylated, favoring aberrant expression of TGF-1 in mesen- chymal cells. TGF-1 activates mesenchymal cells to become myofibroblasts, which perpetuate EndMT and activation of mesenchymal cells through continued induction of TGF-1 and aberrant expression of extracellular matrix, including proteoglycans and collagen.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Derivative Assay, Control, Expressing, Activation Assay

Journal: Nature communications

Article Title: SCARB2 drives hepatocellular carcinoma tumor initiating cells via enhanced MYC transcriptional activity.

doi: 10.1038/s41467-023-41593-z

Figure Lengend Snippet: Fig. 6 | PMB binds with SCARB2 to suppress HCC by decreasing HDAC3- mediated MYC acetylation and MYC transcriptional activity. a–b Virtual screening of FDA-approved drugs to identify small molecules binding with SCARB2 (a), top 10 hits are listed (b). c The kinetics of the SCARB2-PMB interaction were determined by surface plasmon resonance (SPR) analysis. d The highest scoring docking model of the SCARB2 and PMB complex is shown. Top: surface of the PMB- SCARB2 complex. Bottom: 3D structure of the PMB (yellow)-SCARB2 complex. e The kinetics of the SCARB2-MYC interaction with or without PMB were deter- mined by SPR. f Structured illumination microscopic (SIM) images of vehicle- or PMB- treated HCCLM3 cells (1 h) stained for MYC and SCARB2. Scale bar, 10 μm. g The effect of PMB on the interaction of MYC and SCARB2 was evaluated by Co-IP assays. Extracts of DMSO and PMB-treated HCCLM3 cells were IP with an anti- SCARB2 Ab. h Representative images of MYC/HDAC3 colocalization foci in HCCLM3 cells before and after PMB treatment. Scale bar, 10 μm. i Effect of PMB on MYC acetylation. Extracts of DMSO and PMB-treated HCCLM3 cells were IP with an anti-MYC Ab. Acetylated MYC was detected by immunoblotting. j HCCLM3 cells

Article Snippet: Subsequently, 30-μl droplets containing the indicated dilutions of antibodies specific for MYC (R&D, AF3696, 1:100), SCARB2 (Abcam, ab176317, 1:100) or HDAC3 (Novus, NB500-126, 1:100) were placed on Parafilm in a dark humidified chamber.

Techniques: Activity Assay, Binding Assay, SPR Assay, Staining, Co-Immunoprecipitation Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: Primers sequences (5′-3′)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: Differential gene and protein expression in K562 and K562/A02. a Total RNA isolated from K562 and K562/A02 was subjected to RNA-sequencing. The Volcano plot of differentially expressed gene level was defined by analysis of variance. b KEGG pathway analyes of differentially expressed genes. c RT-PCR analysis showed differential gene expression in HL60 and HL60/ADR. d Western blot analysis shows protein expression of differential gene in HL60 and HL60/ADR. e HDAC3 expression of relapsed patients measured with RT-PCR was compared to that of de novo counterparts. f The HDAC3 expression of relapsed samples from the same patients measured with RT-PCR was compared to that of its de novo leukemia samples. g Compared with K562 cells, the HDAC3-AKT-P21-CDK2 signaling pathway was activated in K562/A02 cells (red represents higher expression). Data represents three independent experiments, and results shown are in the format, mean ± S.D. (NS: P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Expressing, Isolation, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: High expression levels of HDAC3 are associated with poorer prognoses . a OS and ( b ) EFS in the primary cohort of 163 AML patients. Multivariable analysis of HDAC3 expression associated with OS( c ) and EFS ( d )

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: Molecular mechanisms of chidamide activity in HL60/ADR and patient-derived anthracycline-resistant AML cells. Anthracycline-resistant AML cells were treated with different concentrations of chidamide. Expression levels of HDAC3, AKT, P21 and CDK2 were measured using RT-PCR in HL60/ADR ( a ) and primary AML cells ( b - c ). d HL60/ADR and ( e ) primary AML cells were treated with chidamide and expression levels of HDAC3, P-AKT, AKT, P21, and CDK2 were measured by Western blot. The expression of HDAC3, AKT, P21, and CDK2 was measured by RT-PCR ( f ) and Western blot ( g ) in HL60/ADR cells after treated with a combination of chidamide and doxorubicin. RT-PCR analysis of HDAC3, AKT,CDK2 and P21 ( h ) and western blot analysis ( i ) in primary AML cells treated with combination therapy. GAPDH was used as the loading control. Data represents three independent experiments, and results are shown in the format, means ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Activity Assay, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: Transfection with HDAC3 shRNA in HL60/ADR cells reduces cell proliferation, increases cell apoptosis, induces cell cycle arrest at G0/G1 phase, and suppresses AKT-P21-CDK2 signaling pathways. a CCK-8 assays were used to assess cell proliferation ability. b Cell apoptosis was analyzed after HDAC3 knockdown by flow cytometry using Annexin-V/PI staining. c is a representative of flow cytometry plot for detection of apoptosis. d-e Cell cycles of HL60/ADR cells were analyzed by flow cytometry after PI staining. f is a representative of flow cytometry for cell cycle detection. g Expression levels of HDAC3, AKT, P21, and CDK2 were measured by RT-PCR. h Expression levels of HDAC3, P-AKT, AKT, P21, and CDK2 were measured by Western blot. GAPDH was used as an internal control. Data represents three independent experiments, and results are shown in the format, mean ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, NS: P > 0.05)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Transfection, shRNA, Protein-Protein interactions, CCK-8 Assay, Knockdown, Flow Cytometry, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: AKT inhibitor reduces cell proliferation, increases cell apoptosis, induces cell cycle arrest at G0/G1 phase, and suppresses the expression of HDAC3, CDK2. HL60/ADR cells were incubated with an AKT inhibitor (MK2206-2HCL). ( a ) CCK-8 assays were used to assess cell proliferation ability. ( b ) Flow cytometry was used to analyze cell apoptosis. c Representative of flow cytometry plot for detection of apoptosis. d-e Cell cycles of HL60/ADR cells were analyzed by flow cytometry after PI staining. f is a representative of flow cytometry for cell cycle detection. g Expression levels of HDAC3, AKT, P21, and CDK2 were measured by RT-PCR. h Expression levels of HDAC3, P-AKT, AKT, P21, and CDK2 were measured by Western blot. GAPDH was used as an internal control. Data represents three independent experiments, results are shown in the format, mean ± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, NS: P > 0.05)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Expressing, Incubation, CCK-8 Assay, Flow Cytometry, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chidamide increases the sensitivity of refractory or relapsed acute myeloid leukemia cells to anthracyclines via regulation of the HDAC3 -AKT-P21-CDK2 signaling pathway

doi: 10.1186/s13046-020-01792-8

Figure Lengend Snippet: Suppression of HDAC3 and AKT are required for chidamide induced anthracycline-resistant AML cells inhibition. HL60/ADR cells were treated with chidamide for 48 h and transfected with overexpressed HDAC3 plasmids or control plasmids. a CCK-8 assays were used to assess cell proliferation ability. b-c Cell cycle detection by flow cytometry. d Detection of apoptosis by flow cytometry. The expression of HDAC3 and AKT was measured by RT-PCR ( e ) and western blot analysis ( f ) HL60/ADR cells were treated with chidamide for 48 h and transfected with overexpressed AKT plasmids or control plasmids. g CCK-8 assays were used to assess cell proliferation ability. h-i Cell cycle detection by flow cytometry. j-k Detection of apoptosis by flow cytometry. The expression of HDAC3 and AKT was measured by RT-PCR (l) and western blot analysis ( m ). n Co-immunoprecipitation of AKT and HDAC3 were performed in 293 T cells. Data represents three independent experiments, data are expressed as mean values± S.D. (* P < 0.05, ** P < 0.01, *** P < 0.001, NS: P > 0.05)

Article Snippet: HDAC3 shRNA plasmid (sc-35,538-SH, Santa Cruz Biotechnology, City, State, Country) was transfected into cells using Superfect reagent (Qiagen) according to the manufacturer’s protocol.

Techniques: Inhibition, Transfection, Control, CCK-8 Assay, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation