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: Aging Cell

Article Title: Hematopoietic progenitor cell liabilities and alarmins S100A8/A9‐related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults

doi: 10.1111/acel.13545

Figure Lengend Snippet: S100A8/A9 high expression levels in CD34+ cells and PB of pre‐frail subjects. (a) RNA sequencing. Heatmaps of modulated transcripts according to CD34 + cell sources (i. bone marrow, ii. peripheral blood). (b) Venn diagram with all deregulated genes (UP = upregulated, DW = downregulated, PB = peripheral blood, BM = bone marrow). (c) Levels of the S100A8/A9 heterodimer in peripheral blood measured by ELISA. Mean values ± SE in the two groups stratified according to the median physical frailty (PF) of 1.5. (d) Heatmap of soluble factors quantified by multiplex ELISA assay. Differences between two groups (PF ≤1.5 or >1.5) were analyzed with unpaired two‐tailed Student’s t test, * p < 0.05 and ** p < 0.01

Article Snippet: PB‐MNCs and BM‐MNCs were assessed for viability by Trypan Blue (SIGMA COD.T8154), and an average of 50 × 10 7 cells was immunomagnetically sorted using Diamond CD34 + HSPC Isolation Kit Human (MILTENYI COD.130‐094‐531).

Techniques: Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Two Tailed Test

Journal: Aging Cell

Article Title: Hematopoietic progenitor cell liabilities and alarmins S100A8/A9‐related inflammaging associate with frailty and predict poor cardiovascular outcomes in older adults

doi: 10.1111/acel.13545

Figure Lengend Snippet: Baseline HSPC and S100A8/A9 levels associates with frailty and predict future MACE. (a) Bar graphs showing the average number of the identified HSPC populations as measured by flow cytometry, and circulating plasma levels of S100A8/A9 in subjects grouped based on the median IFi value of 14.1 ( N = 52 per group). Values are means ± standard deviation. (b) Bar graphs show the abundance of HSPC phenotypes and S100A8/A9 levels in the two groups. (c) Survival free from MACE curves unadjusted (upper panels) and adjusted for age, frailty index and S100A8/A9 (*) or CD34 + CD133 + HSPCs (#), respectively (lower panels). Significance level ( p ‐values) was assessed through Cox proportional hazard model

Article Snippet: PB‐MNCs and BM‐MNCs were assessed for viability by Trypan Blue (SIGMA COD.T8154), and an average of 50 × 10 7 cells was immunomagnetically sorted using Diamond CD34 + HSPC Isolation Kit Human (MILTENYI COD.130‐094‐531).

Techniques: Flow Cytometry, Clinical Proteomics, Standard Deviation

Journal: Science translational medicine

Article Title: Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag

doi: 10.1126/scitranslmed.aas9563

Figure Lengend Snippet: (A) Scheme showing isolation of human HSCs for colony forming assays. (B-C) Megakaryocyte (B) and erythroid (C) colony formation in collagen-based culture system (n=4). (D) Granulocytic colony formation in methylcellulose semi-solid medium (n=7). CFU: colony-forming unit; MK: megakaryocyte colonies; BFU-E: erythroid colonies; CFU-G: granulocyte colonies. Representative morphology for each type of colony is also shown. Scale bars indicate 100 μm. (E) Colonies from primary methylcellulose cultures were serially replated bi-weekly (n=7). Results are represented as fold change of colonies compared to non-treated (NT) vehicle control (water). (F) Fold change of % dividing HSCs compared to vehicle-treated control 24 hours after treatment. % dividing HSCs is expressed as the number of HSCs with at least one completed division out of total HSCs scored. (G) Representative FACS plots of Ki-67 and Hoechst staining of HSCs (Lin-CD34+CD38-CD49f+) exposed to rhTPO and EP at low and high concentrations for 24 hours. (H) Quantification of % cells in G0 versus G1 of cell cycle after stimulation with TPO, EP-lo, and EP-hi for 24 hours in liquid culture compared to vehicle control (NT). rHTPO: 100 ng/ml of recombinant human thrombopoietin, EP-lo: 3 μg/ml of eltrombopag, EP-hi: 10 μg/ml of eltrombopag. n = 4. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Paired Student’s t-test).

Article Snippet: Mononuclear cells were further enriched for CD34 + cells by immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Isolation, Control, Staining, Recombinant

Journal: Science translational medicine

Article Title: Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag

doi: 10.1126/scitranslmed.aas9563

Figure Lengend Snippet: (A-C) HSC frequency in healthy donors enumerated by the LTC-IC assay. Left panel: LTC-IC frequency in each individual donor (n=4). Right panel: scatter plot showing LTC-IC frequency of CD34+ cells in NT versus those treated with iron chelators deferoxamine (10 μM DFO, A), deferasirox (5 μM DFX, B), or EP with or without rescue (C). (D) Representative images of LTC-IC assay showing limiting dilutions of CD34+ cells from healthy donors treated ex vivo with either vehicle (NT) or EP with or without rescue (EP+FAC). (E) FACS analysis of HSCs (Lin- CD34+ CD38-CD49f+) in ITP patients treated with either romiplostim (control) or EP. (F-G) Quantification of phenotypic HSCs within bone marrow mononuclear cell (BMMNC, F) and CD34+ CD38- (G) compartments. (H) Representative images of LTC-IC assay showing CD34+ cells from ITP patients treated in vivo with romiplostim (control) or EP. (I) HSC frequency in ITP patients treated in vivo with romiplostim (n = 2) or EP (n = 4). Scale bars indicate 1 mm. LTC-IC assay. HSC frequencies were estimated by Poisson statistics using ELDA. If not specified otherwise, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Paired Student’s t-test).

Article Snippet: Mononuclear cells were further enriched for CD34 + cells by immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Ex Vivo, Control, In Vivo

Journal: Science translational medicine

Article Title: Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag

doi: 10.1126/scitranslmed.aas9563

Figure Lengend Snippet: (A) Serial colony-forming capacity of Mpl−/− HSCs treated with 10 μg/ml EP compared to NT (water, n=4). (B) LTC-IC assay of Mpl−/− LSK cells after ex vivo treatment with vehicle control (NT) or EP. Bar plot (left panel) showing the fold change of LTC-IC frequency compared to NT, estimated for each of the individual mice (n=4, paired t-test). Scatter plot (right panel) showing LTC-IC frequency for EP and NT. (C) Scheme showing in vivo treatment schedule of Mpl−/− mice. Mice were treated with EP daily for 28 days, followed by immunophenotypic and functional assessment of HSC frequency. (D) Frequency of phenotypic HSCs within the bone marrow after EP (n = 14 mice) or vehicle treatment (NT, n = 12 mice). *p < 0.05 (unpaired t-test) (E-F) Representative images (E) and fold change of stem cell frequency (F) assessed by LTC-IC assay for Mpl−/− LSK cells. Scale bars indicate 1 mm. (G) HSC frequency in EP-treated mice compared to control determined by limiting dilution transplantation. Results are pooled from 5 independent experiments (n=74 recipient mice for NT, and 70 for EP). If not specified otherwise, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Paired Student’s t-test).

Article Snippet: Mononuclear cells were further enriched for CD34 + cells by immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Ex Vivo, Control, In Vivo, Functional Assay, Transplantation Assay