Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-1 was located on the upstream of miR-23a∼27a∼24-2 cluster and activated its expression during erythropoiesis. ( A ) Q-PCR analysis of the primary transcript of the miR-23a∼27a∼24-2 cluster in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( B ) Q-PCR analysis of the mature transcripts of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation caused by hemin treatment for 0, 24, 48 and 72 h. ( C ) Northern blot analysis of miR-27a, miR-24 and miR-23a in K562s undergoing erythroid differentiation. U6 snRNA was used as a loading control. ( D ) Q-PCR analysis of mature miR-27a and miR-24 expression in CD34+ HPCs undergoing day 4, 7, 11, 15 and 18 of E culture. ( E ) Representation of the human −603 bp miR-23a∼27a∼24 cluster promoter fragments. ( F ) ChIP-PCR and ChIP-qPCR analysis of the GATA-1 hit on the −557 site at the miR-23a∼27a∼24 cluster promoter in K562s. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01. ( G ) Immunoblot analysis of GATA-1 expression in K562s treated with siRNAs specific to GATA-1 (si_GATA-1) for 48 h or K562s transfected with a construct overexpressing GATA-1 (over_GATA-1) for 48 h, respectively. ( H ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (G). ( I ) Immunoblot analysis of GATA-1 expression in K562s undergoing erythroid differentiation. ( J ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( K ) Functional activity of GATA-1 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Northern Blot, Control, ChIP-qPCR, Standard Deviation, Western Blot, Transfection, Construct, Functional Assay, Activity Assay, Mutagenesis, Luciferase

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: MiR-27a and miR-24 promoted erythroid differentiation in CD34+ HPCs. ( A ) Monitoring of the GFP + population (left panel) and the CD235a stained GFP + fraction (medium panel) of Lenti-miRNA-transduced CD34+ HPCs on day 15 of E culture. The morphology (May-Grunwald Giemsa staining) of CD34+ HPCs derivatives on day 15 is shown in the right panel. A 400X magnification of a representative field is shown. ( B ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. Percentages of basophilic (Bas), polychromatophilic (Pol), orthochromatic (Ort) erythroblasts and erythrocytes (Ery) were determined by May-Grunwald/Giemsa staining of cytospin preparations. ( C ) Q-PCR analysis of gamma-globin mRNA expression in CD34+ HPCs transduced with Lenti-27a, Lenti-24 or Lenti-GFP in E culture at the indicated time. ( D ) A comparison of the erythroid colony-forming capacity (CFU-E, BFU-E) of CD34+ HPCs transduced with Lenti-miRNAs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) FACS monitoring of CD34+ HPCs transduced with Zip-miRNA or Zip-GFP as described in (A). ( F ) Detection of the erythroid differentiation degree of CD34+ HPCs transduced with Zip-27a, Zip-24 or Zip-GFP as described in (B). ( G ) Detection of gamma-globin mRNA level in CD34+ HPCs transduced with Zip-miRNA. ( H ) Colony-forming assay of CD34+ HPCs transduced with Zip-miRNA as described in (D).

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Staining, Transduction, Expressing, Comparison, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: GATA-2 was post-transcriptionally regulated by miR-27a and miR-24 during erythropoiesis. ( A ) A computer prediction of conserved and mutated binding sites within the 3′ UTR of GATA-2 mRNA for miR-27a and miR-24. ( B ) Relative luciferase activity of the indicated GATA-2 reporter constructs. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( C ) Immunoblot analysis of GATA-2 in K562s transfected with scramble or miRNA mimics (miR-27a, miR-24) or inhibitors (Anti-27a, Anti-24). ( D ) Immunoblot analysis of GATA-2 in CD34+ HPCs transduced with Lenti-GFP control and lentivirus expressing miR-27a or miR-24 (Lenti-27a or Lenti-24). ( E, F ) ‘Rescue’ assays for miRNAs and GATA-2 during erythroid differentiation. Immunoblot analysis of GATA-2 in K562s treated with scramble or Anti-27a/24 (E) for 24 h. These cells were subsequently treated for another 24 h with control siRNAs or siRNAs specific to GATA-2 and were then treated with hemin treatment for 0, 48 and 72 h. (F) FACS analysis of K562s stained for CD71 and CD235a expression after 48 h of hemin induction as described earlier in the text.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Binding Assay, Luciferase, Activity Assay, Construct, Standard Deviation, Western Blot, Transfection, Transduction, Control, Expressing, Staining

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: The GATA switch regulated miR-27a and miR-24 expression. ( A ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1, GATA-2 and Pol II occupancy at the −557 site in hemin-treated K562s at 0 and 48 h. ChIP-q-PCR results are shown as fold enrichment compared with input. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( B ) Immunoblot analysis of GATA-1 and GATA-2 expression in K562s undergoing erythroid differentiation for 0, 24, 48 and 72 h. ( C ) Functional activity of GATA-1 and GATA-2 on the wild-type GATA site (WT) or mutant site (MUT) of the miR-23a∼27a∼24 promoter in a luciferase reporter analysis. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( D ) Immunoblot analysis of GATA-2 in K562s treated with siRNAs specific to GATA-2 (si_GATA-2) for 48 h or K562s transfected with a construct overexpressing GATA-2 (over_GATA-2) for 48 h, respectively. ( E ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in K562s treated as described in (D). ( F ) ChIP-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with control siRNAs or siRNAs specific to GATA-1/GATA-2 or K562s transfected with empty pcDNA3.1 vectors or pcDNA3.1_GATA-1/GATA-2. ( G ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in CD34+ HPCs of E culture. ( H ) Q-PCR analysis of Pri-27a-24 abundance in CD34+ HPCs of E culture (Top panel). Error bars represent the standard deviation obtained from three independent experiments. An immunoblot analysis of GATA-1 and GATA-2 expression in CD34+ HPCs of E culture (Bottom panel). ( I ) Q-PCR (left panel) and immunoblot (right panel) analysis of GATA-1 and GATA-2 expression in CD34+ HPCs transduced with lentivirus-expressing siRNAs against GATA-1 or control on day 11 of E culture. Error bars represent standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( J ) Q-PCR analysis of Pri-27a∼24, mature miR-27a and miR-24 expression in CD34+ HPCs treated as described in (I). Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Expressing, Standard Deviation, Western Blot, Functional Assay, Activity Assay, Mutagenesis, Luciferase, Transfection, Construct, Control, Transduction

Journal: Nucleic Acids Research

Article Title: A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis

doi: 10.1093/nar/gkt848

Figure Lengend Snippet: A regulatory circuit involving GATA-1, GATA-2 and miR-27a/24 in erythropoiesis. ( A ) A schematic representation of the regulatory circuit comprised GATA-1, GATA-2, miR-27a and miR-24 in erythroid differentiation. ( B , C ) ChIP-PCR and ChIP-q-PCR analysis of GATA-1 and GATA-2 occupancy at the −557 site in K562s transfected with scramble or miRNA mimics and inhibitors (B, r miR-27a and C, miR-24). ( D ) Q-PCR analysis of Pri-27a∼24 abundance in K562s treated as described in (B) and (C). Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05; ** P < 0.01. ( E ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Lenti-miR-27a/24 or Lenti-GFP control for 11 days. Error bars represent the standard deviation obtained from three independent experiments. * P < 0.05. ( F ) Q-PCR analysis of Pri-27a∼24 abundance in CD34+ HPCs transduced with Zip-miRNA or Zip-GFP for 11 days. Error bars represent the standard deviation obtained from three independent experiments. ** P < 0.01.

Article Snippet: CD34+ cells were enriched from mononuclear cells through positive immunomagnetic selection (CD34 MultiSort kit, Miltenyi Biotec, Bergisch-Glad-bach, Germany).

Techniques: Transfection, Standard Deviation, Transduction, Control

Journal:

Article Title: CUL-4A stimulates ubiquitylation and degradation of the HOXA9 homeodomain protein

doi: 10.1093/emboj/cdg577

Figure Lengend Snippet: Fig. 6. CUL-4A ablates the HOXA9-induced differentiation block of 32Dcl3 myeloid progenitor cells. (A) Western blotting to detect stable expression of HOXA9 and MYC-CUL-4A in 100 µg of 32Dcl3, 32D-HOXA9, 32D-HOXA9/pcDNA3 and 32D-HOXA9/MYC-CUL-4A (clones 6-16 and 7-1) extracts using antibodies against HOXA9, MYC tag or β-actin. (B) Wright–Giemsa staining of the indicated 32Dcl3 cell lines grown in the presence of 5 ng/ml of IL-3, or induced to differentiate with 20 ng/ml of G-CSF for 8 days. Hemocytograms of the resulting 32Dcl3 cell lines are indicated on the right. The percentages of myoblast, intermediate and granulocytic cells were obtained by counting a total of 500 cells in each experiment, and data are presented as mean ± SD from four independent experiments. (C) The indicated 32Dcl3 cell lines were either untreated or treated with 20 ng/ml of G-CSF for 8 days, and analyzed by flow cytometry for expression of MAC1 (mCD11b) as a marker for granulocytic differentiation. Histograms showed the quantitation of MAC1–FITC+ staining and M1 values in each column indicate the percentages of differentiated (MAC1-positive) 32Dcl3 cells.

Article Snippet: The mononuclear cells at the interphase were collected, and were used to purify CD34 + cells using the immunomagnetic MACS CD34 Isolation kit (Miltenyi Biotec Inc.) according to the manufacturer’s instructions.

Techniques: Blocking Assay, Western Blot, Expressing, Clone Assay, Staining, Flow Cytometry, Marker, Quantitation Assay

Journal:

Article Title: CUL-4A stimulates ubiquitylation and degradation of the HOXA9 homeodomain protein

doi: 10.1093/emboj/cdg577

Figure Lengend Snippet: Fig. 2. CUL-4A is expressed in primitive and differentiated hematopoietic cells. (A) An indirect immunofluorescence assay was carried out on the indicated cells using the anti-CUL-4A polyclonal antibody and secondary antibody conjugated to Cy3 (top). The same cells were also stained with DAPI and photographed under phase contrast lenses (bottom). (B) Subcellular localization of CUL-4A in primary CD34+ hematopoietic stem/progenitor cells by immunofluorescent confocal microscopy. CD34+ cells were also stained with the TO-PRO-3 dye for visualization of nuclei.

Article Snippet: The mononuclear cells at the interphase were collected, and were used to purify CD34 + cells using the immunomagnetic MACS CD34 Isolation kit (Miltenyi Biotec Inc.) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Staining, Confocal Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats

doi: 10.1172/JCI58789

Figure Lengend Snippet: LSECs were isolated by elutriation, and the CD133+ fraction was obtained by immunomagnetic separation to obtain resident SPCs. (A) Staining for CD45 (FITC; green). (B) Staining for CD31 (Alexa Fluor 405; blue). (C) The merged image of A and B demonstrates that these CD133+ cells are also CD45+CD31+. Original magnification, ×100. (D) Scanning EM demonstrates the resident SPCs have fenestration organized in sieve plates characteristic of LSECs. Scale bar: 5 μm.

Article Snippet: Microbeads from the following kits were used: CD133 Cell Isolation Kit and Anti-FITC MultiSort Kit (Miltenyi Biotec).

Techniques: Isolation, Immunomagnetic Separation, Staining

Journal: The Journal of Clinical Investigation

Article Title: Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats

doi: 10.1172/JCI58789

Figure Lengend Snippet: Resident SPCs (CD133+ fraction of LSECs), LSECs with removal of resident SPCs (fraction of LSECs depleted of CD133+ cells), periportal LSECs (CD45bright), and centrilobular LSECs (CD45– LSECs) were stained for VEGF-receptor 1 (VEGF-R1) and VEGF-R2. The third column shows a merged image. Original magnification, ×100.

Article Snippet: Microbeads from the following kits were used: CD133 Cell Isolation Kit and Anti-FITC MultiSort Kit (Miltenyi Biotec).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats

doi: 10.1172/JCI58789

Figure Lengend Snippet: Two months after neonates were injected with BrdU, LSECs were isolated by elutriation, and the CD133+ fraction was isolated by immunomagnetic separation to obtain resident SPCs and LRCs. (A) CD31 staining of resident SPCs and LRCs. (B) CD45 staining of resident SPCs and LRCs. (C) BrdU+ staining indicates LRCs (white arrows). (D) Differential interference contrast (DIC) demonstrates all cells in the field. (E) The merged image shows CD133+ cells that are CD31+CD45+BrdU+ LRCs (white), indicated by white arrows. Original magnification, ×100.

Article Snippet: Microbeads from the following kits were used: CD133 Cell Isolation Kit and Anti-FITC MultiSort Kit (Miltenyi Biotec).

Techniques: Injection, Isolation, Immunomagnetic Separation, Staining, BrdU Staining

Journal: The Journal of Clinical Investigation

Article Title: Liver sinusoidal endothelial cell progenitor cells promote liver regeneration in rats

doi: 10.1172/JCI58789

Figure Lengend Snippet: LSECs were isolated on day 3 after PHx from wild-type rats that had been transplanted with BM from Lew-Tg(CAG-EGFP)ys rats. (A) Hgf mRNA expression was measured by real-time PCR in GFP+ (BM-derived) and GFP– LSECs. n = 3; ***P < 0.001. (B) HGF protein expression was determined by immunoblot and (C) quantified by densitometry in control LSECs (isolated from nonoperated rats) and in GFP+ and GFP– LSECs isolated on day 3 after PHx (n = 3). *P < 0.05, **P < 0.01 compared with GFP– LSECs after PHx. (D) CD133+ cells from the LSEC fraction were immunostained for PCNA and sorted by FACS for GFP. n = 3; ***P < 0.0001 comparison of the percentage PCNA+ cells in the GFP+ and GFP– fractions.

Article Snippet: Microbeads from the following kits were used: CD133 Cell Isolation Kit and Anti-FITC MultiSort Kit (Miltenyi Biotec).

Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Control, Comparison