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

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Insights Into a Chlamydia pneumoniae -Specific Gene Cluster of Membrane Binding Proteins

doi: 10.3389/fcimb.2020.565808

Figure Lengend Snippet: The Mbp1 point mutant (mut) shows reduced association with PtdIns(3)P, Rab11, and Rab14-positive membranes. (A) Schematic representation of wild type (Mbp1) and mutant (Mbp1mut) amino acid sequences and their underlying predicted secondary structures using https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html . H = α-helix E = β-sheet. The gray box displays the transmembrane domain predicted with http://www.cbs.dtu.dk/services/TMHMM/ . Asterisks mark the exchanged amino acids in Mbp1mut. (B) Representative confocal images of HEp-2 cells coexpressing Mbp1 or Mbp1mut proteins fused to GFP and 2xFYVE-mCherry marking PtdIns(3)P. The white box is enlarged in insets and white arrows indicate colocalization of Mbp1 or Mbp1mut with PdtIns(3)-positive vesicles, pink arrows indicate Mbp1 or Mbp1mut vesicles showing no colocalization. Bar: 5 μm, Insets 1 μm. (C–E) Quantification of colocalization of Mbp1 or Mbp1mut with PdtIns(3) (C) , with Rab11 (D) , and Rab14 (E) . Large images of transfected cells were generated and 20 cotransfected cells of 3 different biological replicates were analyzed for colocaliaztion using ImageJ ( n = 3). *** P ≤ 0.001, ** P ≤ 0.01. (F) Membrane lipid strip assay in which lipid strips (lipid positions are shown in the scheme) were incubated with 1 μg/ml His-tagged GST, Mbp1 and Mbp1mut protein. After extensive washing of the membrane, protein binding to the indicated lipids was detected by using an anti-His antibody in combination with an alkaline phosphatase coupled anti-mouse antibody. Immunoblot is representative for two biological replicates ( n = 2).

Article Snippet: Secondary anti-rabbit/mouse antibodies coupled to AP conjugate for immunoblot analysis were obtained from Promega (1:7500) (Fitchburg, Wisconsin, USA).

Techniques: Mutagenesis, Transfection, Generated, Membrane, Stripping Membranes, Incubation, Protein Binding, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Insights Into a Chlamydia pneumoniae -Specific Gene Cluster of Membrane Binding Proteins

doi: 10.3389/fcimb.2020.565808

Figure Lengend Snippet: Mbp11 and Mbp4 are expressed mid-/late in the infection cycle, colocalize with Momp and show a solubilization pattern similar to adhesins and type III secreted proteins. (A–D) Confocal images of mid-to-late Cpn inclusions (24 hpi; 48 hpi) of cells infected with an MOI = 1 (A,B) or of adhering Cpn EBs at 15 min pi (MOI = 10) (C,D) stained with Momp visualized with anti-mouse Alexa594 and specific antibodies for Mbp1 (A,C) , Mbp4 (B,D) , visualized with anti-rabbit Alexa488. DNA was stained with Dapi. White boxes are shown in insets, white arrows indicate colocalization of Mbp1/Mbp4 with Momp at 15 min, 24 and 48 hpi, respectively. (A,B) Bar: 1 μm (15 min pi, 24 hpi and insets). Bar: 10 μm (48 hpi). The images are representative for three different biological replicates ( n = 3). (E,F) Solubilization assay of proteins from purified Cpn EBs following exposure to either PBS, 1% Triton X-100, 2% Sarkosyl, or 1% NP40 + 50 mM DTT for 1 h at 37°C. (E) Model of the Cpn EB indicating the localization of the different tested proteins. The type III secretion needle (T3SS) is shown in black and the outer (OM) and inner (IM) membranes are represented by black circles. The chlamydial outer membrane complex (cOMC) is shown schematically within the gray box. Disulfide bonds are depicted in red. The arrows in different colors indicate the detergents used in the assay. (F) Immunoblot analysis of Cpn EBs treated with PBS alone or in combination with different detergents. Samples were divided into pellet ( P ) and supernatant ( S ) fractions by high speed centrifugation and analyzed by SDS/PAGE using specific antibodies against the analyzed protein. The immunoblot is representative for two biological replicates ( n = 2).

Article Snippet: Secondary anti-rabbit/mouse antibodies coupled to AP conjugate for immunoblot analysis were obtained from Promega (1:7500) (Fitchburg, Wisconsin, USA).

Techniques: Infection, Staining, Purification, Membrane, Western Blot, Centrifugation, SDS Page

Journal:

Article Title: Elimination of Protease Activity Restores Efficient Virion Production to a Human Immunodeficiency Virus Type 1 Nucleocapsid Deletion Mutant

doi: 10.1128/JVI.77.10.5547-5556.2003

Figure Lengend Snippet: Equilibrium density gradient centrifugation of viruses. Profiles of wild-type NL4-3, DelNC, and DelNC/PRR57G viruses subjected to centrifugation through 10 to 50% (wt/vol) sucrose gradients are presented. Amounts of virions detected as measured by RT activity ([3H]TMP incorporation) or by scanning densitometry (pixel gray-scale density) from a p6Gag immunoblot of selected fractions are reported on the y axis versus density of sucrose on the x axis. Virions analyzed are identified to the left of the respective graphs.

Article Snippet: For gradients analyzing DelNC/PR R57G , virions in fractions were detected by p6 Gag immunoblot analysis and relative levels of virions were quantitated by scanning densitometry of the resulting bands with Scion Image for Windows version 4.0.2 (Scion Corp., Frederick, Md.).

Techniques: Gradient Centrifugation, Centrifugation, Activity Assay, Western Blot

Journal: Journal of Experimental Botany

Article Title: Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C 4 grasses

doi: 10.1093/jxb/ery129

Figure Lengend Snippet: Immunoblot analysis of photosynthetic enzymes. Immunoblot analysis for the photosynthetic proteins Rubisco, PEPC, PEP-CK, and NADP-ME extracted from leaves of eight C 4 grasses belonging to three biochemical subtypes in control (C) or shade (S) environments. Loaded volumes varied between 4 μl and 15 μl in order to normalize the protein content to a common leaf area. Because of the small gel size, a limited number of samples (8–9) was loaded on an individual gel. Finally, all immunoblots of the studied protein and species were arranged in a composite figure. For uniform visualization, gamma settings of individual images were adjusted. A protein ladder was used for individual immunoblots; for simplicity, band size is referred to numerically.

Article Snippet: The reactive antiserum was the antigen purified for use in immunoblot analysis (GL Biochem).

Techniques: Western Blot, Control