Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Temporal changes in expression of the C/EBP-ϵ isoforms during eosinophilopoiesis. CD34+ progenitors were differentiated to the eosinophil lineage by suspension culture in SCF, IL-3, IL-5, GM-CSF, and Flt3-L for 3 days, followed by only IL-3 and IL-5 thereafter. The cells were maintained at 0.5 × 106 cells/mL, total and eosinophil counts determined every 3 to 4 days, and total RNA for RT-PCR and total protein for Western blotting prepared from 1 × 106 cells. Cell proliferation (A) and the percentage of eosinophils (B) developing in the cultures based on differential cell counts using Fast Green/Neutral Red staining to distinguish secondary granule formation is shown. Semiquantitative RT-PCR was performed using α-32P-dCTP and C/EBP-ϵ isoform selective primers, with β2M amplified as the internal control for mRNA (cDNA) input (C). C/EBP-ϵ isoform protein expression was analyzed by Western blotting of whole cell lysates using a combination of anti–C/EBP-ϵ C-terminal (C-22; SC-158) and N-terminal (H-75; SC-25770) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), compared with GAPDH expression as the loading control (D). Representative results from 2 independent experiments are shown.

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Amplification

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Effects of the C/EBP-ϵ isoforms on myeloid and erythroid lineage colony formation. CD34+ CB progenitors were transduced with retroviral vectors encoding the C/EBP-ϵ isoforms and after 72 hours of transduction, CD34+GFP+ cells were sorted by FACS and cultured in semisolid Collagen Cult (StemCell Technologies) colony assays using cytokines to drive differentiation of mixed myeloid lineages excluding erythroid (A: culture in SCF, GM-CSF, IL-3, and IL-5), or both mixed myeloid and erythroid lineages (B: culture with SCF, GM-CSF, IL-3, IL-5, and EPO). The mean (± SEM) number of granulocyte-macrophage colony-forming unit (CFU-GM), granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit (CFU-GEMM), and BFU-E colonies that developed from 750 plated CD34+GFP+ transduced progenitors is plotted for 3 or 4 independent experiments performed in duplicate or triplicate. Statistically significant differences of interest are shown (brackets and P values) for comparisons using 1-way analysis of variance (ANOVA) and least significant difference (LSD).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction, Cell Culture

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Effects of the C/EBP-ϵ isoforms on eosinophil, erythroid, and neutrophil differentiation. CD34+ CB progenitors were transduced with retroviral vectors encoding each of the C/EBP-ϵ isoforms. After 72 hours of transduction, CD34+GFP+ cells were sorted by FACS, plated in Collagen Cult colony assays, and the cells induced to differentiate toward the eosinophil (A: culture in SCF, IL-3, and IL-5), neutrophil (B: culture in SCF, IL-3, and G-CSF), and erythroid (C: culture in SCF, IL-3, and EPO) lineages. The mean (± SD) number of GM, eosinophil, and erythroid colonies that developed from 750 plated CD34+GFP+ transduced progenitors is plotted for 3 independent experiments performed in duplicate or triplicate. The effect of the C/EBP-ϵ isoforms on plating efficiency is shown in panel D as the total number of hematopoietic colonies (myeloid + erythroid) developed from 500 plated CD34+GFP+ progenitors for each of the cytokine cocktails used to induce neutrophil (G-CSF), eosinophil (IL-5), and erythroid (EPO) colony formation. Statistically significant differences are shown for comparisons using 1-way ANOVA and LSD (*P ≤ .05, ***P ≤ .001).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: C/EBP-ϵ32/30 enhancement of eosinophil differentiation does not require IL-5. CD34+ CB progenitors were transduced 3 times over a period of 72 hours with the retroviral vector encoding the C/EBP-ϵ32 activator isoform or empty vector control. CD34+GFP+ cells were sorted by FACS, and either plated in Collagen Cult colony assay media containing SCF and IL-3, and the cells allowed to differentiate for 14 days without (A) or with (B) IL-5 added to drive eosinophil colony formation. Eosinophil and GM colonies were enumerated using staining with Fast Green/Neutral Red and May-Grünwald Giemsa, respectively. The mean (± SD) number of CFU-Eos and CFU-GM colonies that developed from 750 CD34+GFP+ transduced progenitors is plotted for triplicate determinations (A,B).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Plasmid Preparation, Colony Assay, Staining

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: The C/EBP-ϵ isoforms differentially induce or inhibit eosinophil gene transcription. Analysis by real-time RT-Q-PCR. CD34+ CB cells were transduced 3 times with the retroviral vectors encoding the 3 C/EBP-ϵ isoforms and empty retroviral vector control over a period of 72 hours, double CD34+GFP+ progenitors sorted by FACS, grown for 14 days in suspension cultures supplemented with IL-3 + IL-5 to drive eosinophil differentiation (as in Figure 6), and total RNA prepared and reverse-transcribed to cDNA. The expression of genes encoding eosinophil secondary granule proteins, including major basic protein-1 (MBP1), eosinophil-derived neurotoxin/ribonuclease-2 (EDN, RNS2), and eosinophil peroxidase (EPX) (A), the soluble and transmembrane alternative RNA splice forms of the eosinophil-specific IL-5 receptor α (IL-5Rα) subunit (B), and GATA-1 and β-globin (C) were analyzed by RT-Q-PCR. The mean cDNA expression levels relative to the expression of the β2M input control in each sample amplified at the same time are plotted (± SD) for 3 independent experiments analyzed in triplicate. Statistically significant differences are shown for comparisons between means using 1-way ANOVA and LSD (*P ≤ .05, ***P ≤ .001).

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Plasmid Preparation, Expressing, Derivative Assay, Amplification

Journal:

Article Title: Human C/EBP-? activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation

doi: 10.1182/blood-2008-02-139741

Figure Lengend Snippet: Transduction of CD34+ progenitors with the C/EBP-ϵ32/30 activator isoforms blocks expression of the C/EBP-ϵ14 repressor isoform. CD34+ cells were transduced for 72 hours with the C/EBP-ϵ32/30 (lane 1) or empty GFP (lane 2) retroviral vectors and grown in suspension cultures supplemented with IL-5 to drive eosinophil differentiation. Total protein and RNA was prepared at 14 days from 106 cells lysed in TRIzol (Invitrogen) and analyzed by Western blotting (A) for expression of the C/EBP-ϵ32/30 and ϵ14 isoforms, or semiquantitative RT-PCR (B) for expression of the C/EBP-ϵ14 repressor isoform. Western blotting for GAPDH was used to control for equal protein loading (A), and PCR for β2M was used for comparison of cDNA inputs (B). Vertical lines have been inserted in panel A to indicate repositioned gel lanes.

Article Snippet: After 72 hours, the cells were stained with anti–CD34-phycoerythrin (PE)–conjugated antibody or IgG1 isotype control (BD PharMingen, San Diego, CA) and sorted by FACS (MoFlo; Dako North America, Carpinteria, CA) based on dual expression of CD34 and GFP.

Techniques: Transduction, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 3 | MPA promotes DSB formation only when combined with Top2 inhibitors that form a Top2-covalent complex. (A) siRNA-mediated knockdown efficiency of Top2α in HeLa cells was analyzed by western blotting. (B) Quantities of DSBs in Top2α-silenced and 3 μM MPA-treated HeLa cells combined with 0.3 μM ETP for 24 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. The representative distribu- tions are shown in the left panel. (C) MPA-induced DSB formation (γH2AX) when combined with ETP was calculated as in the left panel at different Top2 expression levels (siCtrl or siTop2A) in (B). (D) Quantities of nuclear-localized Top2α in 3 μM MPA-treated HeLa cells combined with 0.3 μM ETP for 8 h were analyzed. Representative distributions are shown. (E) Quantities of DSBs in3 μM MPA-treated HeLa cells combined with various Top2 inhibitors, including 0.3 μM ETP, 0.03 μM DOX, 20 μM merbarone (MER), 1 μM ICRF-193 (ICRF), and 10 μM novobiocin (NOVO) for 24 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. Data represent means ± SD of at least three independent experiments.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Knockdown, Western Blot, Flow Cytometry, Expressing

Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 4 | MPA promotes DSB formation in combination with ETP in the vicinity of promoter regions. (A) Location of DSB formation in 10 μM MPA-treated HeLa cells combined with 1 μM ETP was detected by BLISS analysis. The feature distribution was shown using ChIPseeker. (B) Quantities of DSBs in 1 μM ETP-treated HeLa cells under 100 μM DRB treatment for 8 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. (C and D) 10 μM MPA-induced DSBs (C) and nuclear-localized Top2α (D) in HeLa cells when combined with 1 μM ETP under 100 μM DRB treatment for 8 h were analyzed using a flow cytometer. (E) RNA synthesis in HeLa cells treated as indicated was analyzed by EU incorporation using a flow cytometer. Cells were treated with 100 μM DRB and 3 μM MPA 1 h before adding EU, and treatment was continued until the assay. Data represent means ± SD of at least three independent experiments. (F) Volcano plot of MPA-modulated gene expression is shown. The genes with |fold change| ≥ 1.3 and p ≤ 0.01 were considered differentially expressed. The pie chart shows the number of up-regulated and down-regulated DEGs. The arrow indicates the most variable gene (GMPR) in the up-DEGs. (G) Peak profiles around the GMPR locus for ETP and ETP + MPA treatment in the BLISS data are shown. #1 and #2 are the results of two independent experiments.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Flow Cytometry, Gene Expression

Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 5 | Steroids increase DSBs when combined with ETP. (A) Quantities of DSBs in steroid-treated HeLa cells combined with 0.3 μM ETP for 24 h were analyzed by evaluating γH2AX intensity using a flow cytometer. All 54 steroids were used at a concentration of 3 μM. (B) Steroid-induced DSBs when combined with ETP were calculated in (A). (C) Schematic model of steroid-induced synergistic effect with ETP is shown. Top2 molecules trapped in DNA by etoposide are partially released under etoposide monotreatment (left part). In contrast, when steroids are combined with etopo- side, steroid-modulated transcriptional activity collides with the trapped Top2 molecules, which are converted to Top2ccs, resulting in DSB forma- tion and cytotoxicity (right part). This figure was created with BioRender.com.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Flow Cytometry, Concentration Assay, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Clinical use of Dieletrophoresis separation for live Adipose derived stem cells

doi: 10.1186/1479-5876-10-99

Figure Lengend Snippet: Schematic drawing of microelectrodes (black rectangles) trapping a stem cell (green sphere) within a DEP field (dotted lines) in the absence of any laminar flow across the array.

Article Snippet: Aliquots of pre- and post-DEP treated cells were analyzed by Guava-PCA flow cytometry using ViaCount(R) (Millipore 4000–0040 LOT 11–0115) reagent or monoclonal anti-human CD34 directly conjugated to phycoerythrin (R&D Systems FAB7227P LOT: ACOG01) according to manufacturer protocol.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Clinical use of Dieletrophoresis separation for live Adipose derived stem cells

doi: 10.1186/1479-5876-10-99

Figure Lengend Snippet: Schematic drawing of `microelectrodes (black rectangles) trapping a stem cell (green sphere) within a DEP field (dotted lines) in the presence of laminar flow (black arrows) across the array. The cell is displaced laterally to the trailing edge of the DEP field.

Article Snippet: Aliquots of pre- and post-DEP treated cells were analyzed by Guava-PCA flow cytometry using ViaCount(R) (Millipore 4000–0040 LOT 11–0115) reagent or monoclonal anti-human CD34 directly conjugated to phycoerythrin (R&D Systems FAB7227P LOT: ACOG01) according to manufacturer protocol.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Clinical use of Dieletrophoresis separation for live Adipose derived stem cells

doi: 10.1186/1479-5876-10-99

Figure Lengend Snippet: Flow cytometry for detection of CD34 cells in DEP isolated SVF (lower panel) versus non-treated SVF (middle panel) and background control (upper panel).

Article Snippet: Aliquots of pre- and post-DEP treated cells were analyzed by Guava-PCA flow cytometry using ViaCount(R) (Millipore 4000–0040 LOT 11–0115) reagent or monoclonal anti-human CD34 directly conjugated to phycoerythrin (R&D Systems FAB7227P LOT: ACOG01) according to manufacturer protocol.

Techniques: Flow Cytometry, Isolation, Control

Journal: Journal of Translational Medicine

Article Title: Clinical use of Dieletrophoresis separation for live Adipose derived stem cells

doi: 10.1186/1479-5876-10-99

Figure Lengend Snippet: CD34 cell counts expressed as a percentage cell population determined by flow cytometry, before and after DEP isolation.

Article Snippet: Aliquots of pre- and post-DEP treated cells were analyzed by Guava-PCA flow cytometry using ViaCount(R) (Millipore 4000–0040 LOT 11–0115) reagent or monoclonal anti-human CD34 directly conjugated to phycoerythrin (R&D Systems FAB7227P LOT: ACOG01) according to manufacturer protocol.

Techniques: Flow Cytometry, Isolation