Journal: Cell reports

Article Title: Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5

doi: 10.1016/j.celrep.2023.113145

Figure Lengend Snippet:

Article Snippet: MYC Epitope Tag Antibody Rabbit Polyclonal , Rockland , Cat#600-401-381; RRID:AB_217927.

Techniques: Control, Recombinant, Affinity Purification, Virus, Plasmid Preparation, Quantitative Proteomics, Over Expression, Knock-Out, Software

Journal: Breast Cancer Research : BCR

Article Title: BHLHE40 confers a pro-survival and pro-metastatic phenotype to breast cancer cells by modulating HBEGF secretion

doi: 10.1186/s13058-018-1046-3

Figure Lengend Snippet: BHLHE40 activates gene expression by sequestering histone deacetylase (HDAC)1 and HDAC2 from genome DNA binding in MDA-MB-231 cells exposed to hypoxia and low glucose (1%O 2 /LG, 4 h). a BHLHE40-knockout (KO) diminished dissociation of HDAC1 and HDAC2 from the promoter region of HBEGF in MDA-MB-231 cells exposed to 1%O 2 /LG (4 h), as determined by chromatin immunoprecipitation (ChIP) followed by qPCR of the HBEGF promoter region (−529 to −372 from the transcription start site). HBEGF promoter binding activity of HDAC1 or HDAC2 was calculated as: (DNA amount in anti-HADC IP complex – DNA amount in control IgG IP complex)/DNA amount in 1% input. * p < 0.05 ( n = 6, 1%O 2 /LG vs. control), ** p < 0.05 ( n = 6, KO vs. EV). b 1%O 2 /LG treatment increased interactions between BHLHE40 and HDAC1/2 in the soluble cellular fraction of MDA-MB-231 empty vector (EV) cells. Protein-protein interaction was detected by reciprocal co-immunoprecipitation (IP)/immunoblotting (IB) analysis. c HDAC inhibition induced expression of BHLHE40 target genes. Cells were exposed to hypoxia (1% O 2 ) or HDAC inhibitors (BRD6688 10 μM or TSA 2 μM) for 24 h. mRNA expression levels were determined by qPCR, normalized to RPL13A, and presented as mean ± SD ( n = 6). * p < 0.05 ( n = 6, treated vs. untreated control cells), one-way ANOVA followed by Tukey’s post-hoc tests

Article Snippet: To detect protein-protein interaction, soluble proteins were extracted using the Pierce IP Lysis Buffer (Thermo Scientific) supplemented with protease inhibitor cocktails and co-immunoprecipitation (CoIP) was performed using the TrueBlot Immunoprecipitation and Western Blot Kit (Rockland Immunochemicals Inc., Limerick, PA, USA).

Techniques: Gene Expression, Histone Deacetylase Assay, Binding Assay, Knock-Out, Chromatin Immunoprecipitation, Activity Assay, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Inhibition, Expressing

Journal:

Article Title: Performance of Commercially Available Enzyme Immunoassays for Detection of Antibodies against Herpes Simplex Virus Type 2 in African Populations

doi: 10.1128/JCM.42.7.2961-2965.2004

Figure Lengend Snippet: Initial selection of HSV-2-specific serological tests

Article Snippet: In 2001, three tests were approved by the U.S. Food and Drug Administration (FDA) for the diagnosis of HSV-2 infection, including the HerpeSelect (Focus Technologies, Inc., Cypress, Calif.), the HSV-1 and HSV-2 IgG differentiation immunoblot (Focus Technologies), and the POCkit HSV-2 (Diagnology, Ltd., Belfast, Northern Ireland).

Techniques: Selection, Purification, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Treatment with DZNep induces apoptosis in a dose- and time-dependent manner and markedly reduces clonogenic survival of AML cells. (A) OCI-AML3 cells were treated with the indicated concentrations of DZNep for 24 hours, then fixed and stained with propidium iodide, and cell-cycle status was determined by flow cytometry. *G0/G1 values significantly different from untreated cells; †S-phase values significantly different from untreated cells; ‡G2/M values significantly different from untreated cells. (B) HL-60 and OCI-AML3 cells were treated with the indicated concentrations of DZNep for 72 hours. Then, the cells were stained with annexin V, and the percentages of apoptotic cells were determined by flow cytometry. Columns represent the mean of 3 independent experiments; bars represent SEM. (C) OCI-AML3 and HL-60 cells were left untreated, or treated with 500 nmol/L DZNep, for 48 and 72 hours. After this, total cell lysates were prepared and immunoblot analysis was performed for PARP. The expression levels of β-actin in the lysates served as the loading control. A vertical line has been inserted to indicate a repositioned gel lane. (D) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 48 hours. After treatment, colony growth in semisolid media was assessed after 7 days. Bar graphs represent the mean percentage values ± SEM of untreated colony growth.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Staining, Flow Cytometry, Western Blot, Expressing

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Treatment with DZNep depletes expression of polycomb group proteins EZH2, SUZ12, and EED in cultured and primary AML cells. (A) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. After this, total cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, and DNMT1. The expression levels of β-actin in the lysates served as the loading control. (B) Primary AML cells were treated with the indicated concentrations of DZNep for 24 hours. At the end of treatment, cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, and DNMT1. The expression levels of β-actin in the lysates served as the loading control. (C) OCI-AML3 cells were treated with BZ and DZNep as indicated for 8 hours. After treatment, cell lysates were prepared and immunoblot analysis was performed for EZH2 and SUZ12. The expression levels of β-actin in the lysates served as the loading control. (D) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 16 hours. Then, total RNA was isolated and quantitative real-time PCR was performed with TaqMan probes for EZH2, SUZ12, and EED. The relative quantity (RQ) of each mRNA was normalized against glyceraldehyde-3-phosphate dehydrogenase expression. (E) HL-60 and OCI-AML3 cells were treated with 2 μM DZNep for 8 and 24 hours. Total RNA was isolated and reverse transcribed with a stem loop primer for hsa-miR-101. After reverse transcription, qPCR for hsa-miR-101 was performed, and expression of hsa-miR-101 was normalized against 18S RNA expression. (F) HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. Chromatin immunoprecipitation was performed with anti-EZH2 antibody. Immunoprecipitated DNA was used for qPCR of the WNT1, CHD1, and HOXA9 promoters. Fold enrichment data are relative to the control and are expressed as a ratio of the cycle threshold for the chromatin immunoprecipitation DNA versus the cycle threshold for the input samples.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Expressing, Cell Culture, Western Blot, Isolation, Real-time Polymerase Chain Reaction, RNA Expression, Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: DZNep treatment depletes trimethylation of K27 on histone H3 and induces the expression of cell-cycle regulatory genes p16, p21, and p27, as well as the cell death regulator FBXO32 in AML cells. (A) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. After this, nuclear extracts were prepared and immunoblot analysis was performed for 3MeK27 histone H3, 3MeK9 histone H3, 3MeK79 histone H3, and 3MeK4 histone H3. The expression levels of histone H3 in the extracts served as the loading control. (B) OCI-AML3 cells were treated with the indicated concentrations of DZNep for 24 hours. After treatment, total RNA was isolated and RT-PCR was performed for FBXO32, p16, p21, and p27. A β-actin–specific reaction and expression levels served to ensure equal loading. (C) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep for 24 hours. After this, total cell lysates were prepared and immunoblot analysis was performed for FBXO32, cyclin E, p16, p21, and p27. The expression levels of β-actin in the lysates served as the loading control. (D) OCI-AML3 cells were transfected with scrambled control or EZH2 siRNA for 48 hours. Then, total RNA was isolated and RT-PCR was performed for EZH2, SUZ12, EED, FBXO32, p16, p21, and p27. A β-actin–specific reaction and expression levels served to ensure equal loading. Alternatively, total cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, FBXO32, p16, p21, p27, and 3MeK27 histone H3. The expression level of β-actin in the lysates served as the loading control.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Cotreatment with PS and DZNep synergistically induces apoptosis of cultured AML cells and significantly prolongs survival of mice implanted with AML cells. (A) OCI-AML3 and HL-60 cells were treated with DZNep and/or PS as indicated for 48 hours. At the conclusion of treatment, cell death was assessed by trypan blue dye uptake in a hemocytometer. Columns represent the mean of 3 independent experiments; bars represent SEM. (B) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep and/or PS for 24 hours. Cell lysates were prepared and immunoblot analysis was performed for PARP cleavage. The levels of β-actin in the lysates served as the loading control. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Analysis of dose-effect relationship for DZNep (100-750 nmol/L) and PS (5-20 nmol/L) for the apoptotic effects after 48 hours of exposure in OCI-AML3 and HL-60 cells was performed according to the median dose-effect method of Chou and Talalay. After this, the CI values were calculated. CI < 1, CI = 1, and CI > 1 represent synergism, additivity, and antagonism of the 2 agents, respectively. (D) Female NOD/SCID mice were injected in the lateral tail vein with HL-60 cells. The cells were allowed to engraft for 7 days before initiation of treatment. Mice were treated intraperitoneally with dimethylsulfoxide, 1 mg/kg DZNep 2 days per week for 2 weeks, and/or 10 mg/kg PS 3 days per week for 4 weeks. n = 7 per group. Survival of the mice in all groups (vehicle, DZNep alone, PS alone, and combination) is represented by Kaplan-Meier plot.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Cell Culture, Western Blot, Injection

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Cotreatment with PS enhances DZNep-mediated depletion of EZH2, SUZ12, and EED protein and induction of p16, p27, and FBXO32 protein in AML cells. (A-B) OCI-AML3 and HL-60 cells were treated with the indicated concentrations of DZNep and PS for 24 hours. Then, total cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, DNMT1, 3MeK27H3, acetyl K27H3, FBXO32, cyclin E, p16, p21, p27, and HOXA9. The expression levels of β-actin in the lysates served as the loading control.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Western Blot, Expressing

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Treatment with DZNep and/or PS induces differentiation of AML cells. (A) Immunoblot analysis of HL-60 and OCI-AML3 cells treated for 48 hours with the indicated concentrations of DZNep and/or PS. The expression levels of β-actin in the lysates served as the loading control. Vertical lines have been inserted to indicate repositioned gel lanes. (B) HL-60 and OCI-AML3 cells were treated with the indicated concentrations of DZNep and/or PS for 48 hours. After this, cells were washed and stained with CD11b antibody, and the percentages of CD11b+ cells were determined by flow cytometry. (C) HL-60 and OCI-AML3 cells were treated with the indicated concentrations of DZNep and/or PS for 72 hours. After treatment, the cells were cytospun onto glass slides, Wright stained, and observed with a microscope to assess cellular morphology.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Western Blot, Expressing, Staining, Flow Cytometry, Microscopy

Journal: Blood

Article Title: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells

doi: 10.1182/blood-2009-03-213496

Figure Lengend Snippet: Cotreatment with DZNep and PS exerts a greater antileukemia effect than either agent alone in primary AML cells. (A) Peripheral blood or bone marrow from 4 patients with AML, CD34+ cells enriched from 4 AML patients, and CD34+ cells from 2 normal donors were treated with the indicated concentrations of DZNep and/or PS for 48 hours. Then, the percentages of nonviable cells for each drug alone or drug combination were determined by trypan blue dye uptake in a hemocytometer. Columns represent the mean of the samples; bars represent SEM. *Values significantly greater (P < .05) than those after treatment with either agent alone at the indicated concentrations in the AML samples. †Values significantly less (P < .05) in normal CD34+ versus leukemia samples for the drug combinations. (B) Primary AML cells were treated with the indicated concentrations of DZNep and/or PS for 24 hours. After treatment, cell lysates were prepared and immunoblot analysis was performed for EZH2, SUZ12, EED, and DNMT1. The expression levels of β-actin in the lysates served as the loading control. (C) CD34+/CD38−/Lin− cells enriched from the bone marrow of AML patients were treated with the indicated concentrations of DZNep and/or PS for 48 hours. The percentages of nonviable cells for each drug alone or drug combination were then determined by trypan blue dye uptake in a hemocytometer. Columns represent the mean of the samples; bars represent SEM. (D) Immunoblot analyses of CD34+ cells from a normal donor treated with the indicated concentrations of DZNep and/or PS for 24 hours. The expression levels of β-actin in the lysates served as the loading control.

Article Snippet: 19 , 31 , 32 Banked, delinked, and deidentified donor peripheral blood CD34 + mononuclear cells that were procured, but not used for engraftment in the recipients, were purified by immunomagnetic beads conjugated with anti-CD34 antibody before utilization in the cell viability assay and immunoblot analysis (StemCell Technologies).

Techniques: Western Blot, Expressing