Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Differentiation of CB-CD34+ cells in a feeder- and serum-free culture system. A, Schematic of culture system. B, Conceptual schema of human T-cell development. CD7, CD5, and CD1a are sequentially expressed on CD34+ cells entering into the thymus (DN cells), and develop into CD4- or CD8-expressing single positive (SP) cells through immature SP (iSP) and immature double-positive (iDP). Finally, mature SP T cells leave thymus and spread to periphery. C, Representative histograms and flow cytometry plots on day 14. Mean ± SEM values are presented for percent of CD7+ and CD7+CD5+ cells. D, The CD7+ (proT1) cell numbers on day 14 were different between groups without AA under ambient air and groups with AA under physioxia. CD7+ cell number showed differences between physioxia and ambient air groups (n = 9, two-way ANOVA, *P < .05, **P < .01). E, CD7+CD5+ (proT2) cell numbers showed differences between ambient air and physioxia groups (n = 9, two-way ANOVA, *P < .05). F, Folds of numbers of CD33+ (myeloid) cells on day 14 (n = 4, *P < .05). Cell numbers were normalized as cell numbers plated into one well (4000 CD34 + HSC/HPCs). Plots are presented as mean ± SEM. H, physioxia; HA, physioxia with AA; N, ambient air (non-physioxia); NA, ambient air with AA; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Expressing, Flow Cytometry

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: HSC/HPC populations in physioxia vs ambient air groups. Cell numbers of (A) HSC/HPC (CD34+CD38−), (B) HSC (CD34+CD38−CD45RA−CD90+), and (C) multipotent progenitor (MPP, CD34+CD38−CD45RA−CD90−) were not different between physioxia and ambient air groups. D, Higher numbers of Lymphoid-primed multipotent progenitors (LMPP, CD34+CD38−CD45RA+CD90lo/−CD10−) and multi-lymphoid progenitors (MLP, CD34+CD38−CD45RA+CD90lo/−D10+) were observed in physioxia groups than in ambient air groups (P = .056) on day 7 (n = 3). E, Gene expression of the Notch1 signaling pathway (Notch1, HES1, DELTEX, TCF7, and GATA3) and PU.1 were not differently expressed between physioxia and ambient air groups at 48 hours (n = 4). Data are expressed as mean ± SEM. H, physioxia; HA, physioxia with AA; N, ambient air; NA, ambient air with AA; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Gene Expression

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Maturation of progenitor T cells on artificial thymic organoid cultures (ATO). A, Experimental schema. B, ATO at week 6. Images of ATOs divided by four or nine pieces at ×10 magnification were taken using inverted microscope (Eclipse Ts2R, Nikon, Tokyo, Japan), then merged those into one images using a large imaging stitching tool with NIS-elements Basic Research software (Nikon). C, Representative flow cytometry plot showing progenitor T cells and T-cell development on ATOs. Week 2 is the timing of placing progenitor cells onto ATOs, and week 6 is the timing for harvesting ATO cells for further analysis. More abundant expression of progenitor T and mature T-cell markers on cells were noted at week 6 than at week 2, with a comparison of cells maintained under ambient air (21% O2) vs under physioxia (5% O2) (n = 3)

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques: Inverted Microscopy, Imaging, Software, Flow Cytometry, Expressing, Comparison

Journal: Stem cells (Dayton, Ohio)

Article Title: Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

doi: 10.1002/stem.3259

Figure Lengend Snippet: Effect of physioxia vs ambient air during the maturation phase in ATOs. A, Experimental schema. B, ATO cell numbers under physioxia (5% O2) were increased more than those under ambient air (21% O2). Cell numbers were normalized by cell numbers placed onto one ATO (7500 progenitors/ATO) and cell numbers plated into one well (4000 CD34+ cells/well) (n = 3, two-way ANOVA, *P < .05). C, Cell numbers of CD7+CD1a+ precursor T cells per 4000 CD34+ cells between physioxia vs ambient air groups in ATOs (n = 3, two-way ANOVA, **P < .01). D-F, Numbers of T-cell subsets per 4000 CD34+ cells between physioxia vs ambient air groups in ATOs on week 6 (n = 3, two-way ANOVA, *P < .05). Data are presented as mean ± SEM. Diff., Differentiation; Mat., Maturation; NS, not significant

Article Snippet: CD34+ cell fractions were further isolated through a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, California).

Techniques:

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: DNA methylation changes during hematopoietic differentiation. ( A ) Matrix showing the number of demethylated CpG sites in each hematopoietic cell subset and demethylated CpG sites shared between distinct hematopoietic cell types. ( B ) Illumina array methylation clustering heatmap of SHEF-1 hESC line hypermethylated genes, demethylated in at least one of the six hematopoietic cell types analyzed: CD34 + HSPCs, neutrophils, B cells, NK cells, CD8 + T cytotoxic cells (CD8 + ) and CD4 + T helper cells (CD4 + ). Methylation levels are indicated as in B. ( C ) Box plots of microarray-based gene expression data (log scale). In each blood cell type, specific demethylated genes exhibited higher expression levels compared to other cell types. P -values are shown. n = number of genes analyzed.

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: DNA Methylation Assay, Methylation, Microarray, Gene Expression, Expressing

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: Promoter methylation and expression levels of hematopoietic genes in CD34 + HSPCs, cells differentiated from CD34 + and iPSCs generated from CD34 + HSPCs. ( A ) High purity sorted CB-derived CD34 + HSPCs (top middle panel) were differentiated in vitro (Diff-CD34) for 14 days in the presence of SCF, GM-CSF, IL3 and EPO. Granulocyte (CFU-G), monocyte (CFU-M), granulo-monocyte (CFU-GM) and erythroid (BFU-E) colony forming units were scored by light microscopy (right panels). Additionally, CB-derived CD34 + HSPCs were induced to travel back in development by generating iPSCs through ectopic expression of Oct4, Klf4, Sox2 and c-Myc (left panel shows a phase contrast image of a CD34-iPSC). The bottom panel shows a scatter plot of remethylated genes in CD34-iPSC (red) ( Supplementary Table S13 ) and demethylated genes in the differentiated CD34 progeny (Diff–CD34) (green) ( Supplementary Table S12 ). ( B ) Promoter methylation and expression of HLA-DR, CD31 and PIK3CD. Methylation and expression for each gene is indicated as in . Expression by flow cytometry of each gene in CD34-iPSC (top panel) and CD34 + HSPCs (bottom panel). For PIK3CD, WB analysis was performed in CD34 + HSPCs, CD34-iPSC and Diff-CD34 (β-actin was used as loading control).

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: Methylation, Expressing, Generated, Derivative Assay, In Vitro, Light Microscopy, Flow Cytometry, Control

Journal: Nucleic Acids Research

Article Title: A promoter DNA demethylation landscape of human hematopoietic differentiation

doi: 10.1093/nar/gkr685

Figure Lengend Snippet: Cartoon depicting the overall methylation levels of hematopoietic genes at different developmental/differentiation stages: hESC, CD34 + HSPCs and mature hematopoietic cell types (undifferentiated stages, purple nuclei; neutrophils, pink nucleus; lymphoid cells, blue nuclei). For each differentiation stage, the heatmap shows the methylation levels of a selected group of hypermethylated genes in hESCs that are demethylated in that cell type ( Supplementary Table S11 ). Names of some key blood genes are mapped at the right of each methylation heatmap.

Article Snippet: The CB-derived CD34 + -enriched fraction (2 × 10 3 cells/cm 2 ) was plated in methycellulose-based medium supplemented with SCF (50 ng/ml), GM-CSF (10 ng/ml), IL-3 (10 ng/ml) and erythropoietin (3 U/ml; Methocult GF H4434; StemCell Technologies).

Techniques: Methylation

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 loss in leukemic progenitors and differential induction of its cell context–independent tumor suppressor activities in quiescent LSCs. A, (left) MIR300 levels in healthy NBM and CML-CP and -BC CD34+ BM cell fractions. Inset shows MIR300 levels in additional CD38-fractionated CD34+ CML-CP BM cells expressed as n-fold difference in CD34+CD38+ compared with CD34+CD38− samples. B, MIR300 levels in untreated and imatinib (24 hours)-treated CD34+ quiescent (CFSEmax) and dividing (Div.1) CFSE-labeled CML and UCB cells. Asterix on CD34+CD38− cell populations (panel 1a) indicate significance between MIR300 levels CD34+CD38− versus CD34+CD38+ cells. Data are shown as mean ± SEM from at least three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Labeling

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 activity in quiescent leukemic stem and progenitor cells. A, Growth (48 hours) and clonogenic potential (CFC) of CpG-scramble- and CpG-miR-300-treated (500 nmol/L) CD34+ CML-BC and UCB cells. B, Effect of CpG-miR-300 and CpG-scramble (500 nmol/L) on spontaneous and IM (18 hours)-induced apoptosis (Annexin V/7-AAD) in CD34+ CML-BC cells (n = 3). Data are reported as mean ± SE (P < 0.01) from three independent experiments inside representative Annexin V/7AAD FACS pseudocolor plots. C, Ki-67/DAPI (left; G0: MIR300 = 46% vs. scr ≅ 10%; G1: MIR300 ≅ 15% vs. scr ≅ 50%; S/G2–M: MIR300 ≅ 4% vs. scr ≅ 27.4%; and sub-G1: MIR300 ≅ 35% vs. scr ≅ 3%) and FUCCI-2BL (right; G1–G0: MIR300 ≅ 40.2% vs. scr ≅ 23.6%; G1–S: MIR300 ≅ 15% vs. scr ≅ 2.85%; S/G2–M: MIR300 ≅ 45% vs. scr ≅ 76.6%) cell-cycle analysis of UBC and Ph+ (primary CD34+ and synchronized LAMA-84) cells exposed to the indicated CpG-ONs. D, Dose-dependent differential regulation of MIR300 antiproliferative and proapoptotic activities on CML qLSC (CFSEmax) and progenitor (Div. 1–2) cell (left) and LTC-IC (right) numbers. Vector transduced and 500 nmol/L CpG-scramble and CpG-anti-miR-300 served as controls. Inset, MIR300 levels in pCDH-MIR300 lentiviral–transduced and 250–500 nmol/L CpG-miR-300–treated Ph+ cells. Data are shown as mean ± SEM from at least three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Cell Cycle Assay, Plasmid Preparation

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: C/EBPβ-dependent MIR300 tumor suppressor antiproliferative activity accounts for BMM-induced LSC entry into quiescence. A, Effect of hypoxia on (i) MIR300 levels in CD34+ CML-BC, and BM-derived primary (hMSCs) and HS-5 MSCs (B); and (ii) MIR300 targets in untreated and CpG-anti-miR-300–treated (500 nmol/L, 48 hours) and CML-BC cells. Inset, effect of hypoxia on CFSE+CD34+ CML-BC proliferation. B, Effect of hMSC and HS-5 conditioned medium (CM) and/or exosomes (50–100 μg/mL) from parental, vector (pZIP), and anti-MIR300 (pZIP-MIR300)–transduced primary hMSCs and/or HS-5 cells on: (i) proliferation (% growth inhibition); (ii) qLSC fraction (CFSEmaxCD34+); and (iii) MIR300 levels in CD34+ CML-BC and LAMA-84 cells. Insets, β-catenin and SET levels in anti-MIR300 (pZip-300)-transduced HS-5 (left); MIR300 in HS-5 Alix+CD63+ exosomes (right). E, Effect of hypoxia on primary MIR300 transcripts (pri-miR-300), C/EBPβ (LAP1, LAP2, and LIP isoforms), BCR-ABL1 expression (αABL) and activity (αPY), and GRB2 levels in CD34+ CML-BC cells. (*): nonspecific band. E, MIR300 promoter/enhancer activity in hypoxia- (48 hours) and normoxia-cultured CML-BC CD34+ cells transduced with pGFP/Luc-based MIR300-reported constructs. p109mut is mutated in the −64 and −46 bp C/EBPβ-binding sites. F, Effect of ectopic C/EBPβ (inset) on mature (MIR300) and primary (pri-miR-300) MIR300 levels in CD34+ CML-BC cells. Data are represented as mean ± SEM for at least three experiments. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Derivative Assay, Plasmid Preparation, Inhibition, Expressing, Cell Culture, Transduction, Construct, Binding Assay

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: MIR300 acts as master PP2A activator and inhibitor of G1–S transition through a dose-dependent target selection mechanism. A, Left:, representative blots show effect of MIR300 on its targets and PP2A activity in UCB and CML-BC CD34+ cells and cell lines exposed to CpG-scramble and CpG-miR-300 (500 nmol/L; 48–72 hours). Right, (top) Dapi/Ki67 cell-cycle analysis of CpG-scramble, -miR-300, and CpG-anti-miR-300 (500 nmol/L; 21 hours)-treated aphidicolin-synchronized K562 cells; (middle) Flag-SET lentiviral constructs with wild-type or a deleted mRNA 3′UTR; (bottom) MIR300-induced downregulation of Flag-SET proteins, and rescue of Ph+ cells from exogenous MIR300-induced growth inhibition (Trypan blue exclusion)/apoptosis (Annexin V+) by Flag-SET cDNAs lacking MIR300-binding site. Similar results were obtained with LAMA-84 cells. B, Hierarchical clustering of statistically significant (P < 0.05 with FDR correction) MIR300 targets using the indicated databases (number of binding sites is indicated in red). Top right, schematic representation of the biological effects of MIR300 dose-dependent target selection activity in qLSCs and leukemic progenitors; (bottom) SET, CDK6, CCND2, and β-actin levels in CpG-MIR300- and CpG-scramble–treated (100–500 nmol/L; 48 hours) CML-BC CD34+ cells.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Selection, Activity Assay, Cell Cycle Assay, Construct, Inhibition, Binding Assay

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: Selective suppression of MIR300 proapoptotic, but not antiproliferative, activity by TUG1 lncRNA in CML quiescent LSCs. A, BMM-generated signals regulating MIR300-TUG1 interplay and its effect on CML LSC survival and quiescence. B, TUG1 levels in CD34+ quiescent stem (CFSEmax) and dividing progenitors (Div.1, 2) and in untreated and imatinib-treated CD34+ CML cells. C, Effect of anti-TGFβ antibody (Ab) and/or hypoxia (1% O2, 48 hours) on TUG1 lncRNA and FoxM1 levels in CD34+ and CD34+CFSEmax CML-BC cells. D, Dose-dependent differential effect of low (100 nmol/L) and high (500 nmol/L) CpG-TUG1-shRNA and CpG-scramble on Ph+ LAMA-84 cell proliferation and survival (Annexin V). E, Effect of anti-TGFβ Ab, TUG1-shRNA, TUG1 RNA, and control (CpG-scramble or empty vector) on recovery of untreated and CpG-scramble, -miR-300, and/or -anti-miR-300 eFluor+CD34+ CML qLSCs (eFluormax) and dividing (Div.1–2) progenitors relative to input. Inset, TUG1 levels in vector, TUG1 shRNA and scramble-shRNA cells. Data are represented as mean ± SEM for at least three experiments. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Activity Assay, Generated, shRNA, Control, Plasmid Preparation

Journal: Blood cancer discovery

Article Title: Persistence of Drug-Resistant Leukemic Stem Cells and Impaired NK Cell Immunity in CML Patients Depend on MIR300 Antiproliferative and PP2A-Activating Functions

doi: 10.1158/0008-5472.BCD-19-0039

Figure Lengend Snippet: Disruption of MIR300-TUG1 interplay and PAD treatment abrogate the BMM-protective effect on survival of CML qLSCs and BCR-ABL1+ leukemia-initiating cells. A, Xenotransplantation protocol of ex vivo–treated CD34+ chronic (CP), accelerated (AP) and blastic phase (BC) CML cells in NRG-SGM3 mice (n = 4 mice/treatment/patient sample). B and C, Analysis of CpG-MIR300-, CpG-TUG1-shRNA-, CpG-TUG1-shRNA+CpG-MIR300-, and CpG-scramble–treated CML cells from BM aspirates at 2–12 (3D plots) and 10 to 20 weeks posttransplant quantitative analysis of CML cells stained with the indicated antibodies. D, Evaluation at 10 to 20 weeks posttransplant of BCR-ABL1 transcripts by qRT-PCR (left) and of % Ph-negative (Ph−) cells by FISH (right) in total and FACS-sorted hCD45+BM cells, respectively. E, Analyses of BM CML cells at 10 to 20 weeks posttransplant: hCD45+ cells (%) in BM (left) and PB (right) of mice transplanted with CML (CP, AP, and BC) and treated with the indicated CpG-ODNs. Age-matched mice served as controls. Error bars, mean ± SEM. F, Effect of 2.5 μmol/L FTY720 or 1 μmol/L imatinib (IM) on CAFC activity (left) and numbers of CFSEmaxAnnexinVneghCD45+CD34+ CML qLSCs derived from CFSE-labeled CD34+ CML-BC cells cocultured for 7 days on BM-derived HS-5 MSC cells (right). Inset, FACS plot shows gating of CFSEmax CML qLSCs. G, Relative number and representative images of CAFC (red arrows) of CFSE-labeled 32D-BCR-ABL cells cocultured with primary mMSCs in the absence or presence of IM (1 μmol/L, 48 hours) or FTY720 (2 μmol/L, 48 hours; n = 5). Inset: CFSEbright fraction of adherent 32D-BCR-ABL cells in medium and cocultured for 4 days with mMSCs. Range values of controls are reported in Supplementary Table S1.

Article Snippet: Flow Cytometry and Cell Sorting CD34 + , CD34 + CD38 − , CD34 + CD38 + fractions were magnetic (CD34 MicroBead Kit; Miltenyi Biotec) and/or FACS (αCD34 APC/PE and αCD38 PE/Cy7 Abs, BD Biosciences) purified (purity: >90%–100%).

Techniques: Disruption, Ex Vivo, shRNA, Staining, Quantitative RT-PCR, Activity Assay, Derivative Assay, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Adaptive from Innate: Human IFN-γ + CD4 + T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults

doi: 10.4049/jimmunol.1700551

Figure Lengend Snippet: CXCL8-producing T cells decline with age in humans and in vivo in humanized mice and are enriched in RTEs. ( A ) CXCL8 production was determined in naive CD4 + T cells (4 h PI + BFA) obtained from 45 individuals (aged between 3 mo and 57 y). ( B ) Irradiated NSG mice were reconstituted with human CD34 + cells (CB derived). The graph shows reconstitution of T cells over time in individual mice (gray lines), with a reciprocal decline in T cell production of CXCL8 (black lines). ( C ) FACS plots show an example of this reciprocal change in one reconstituted NSG mouse; percentage of cells expressing CD3 within human CD45 + cells (upper panels) and percentage of cells expressing CXCL8 among CD3 T cells (lower panels; PI + BFA or BFA alone for 4 h). CD4 + T cells from adults ( D and F ) or children aged 1–12 y ( E ) were activated with PI (4 h), and cytokine production was determined by intracellular staining. Individual (cytokine + ) subsets were then sorted, and TREC content was determined by qPCR. Results are shown as TREC levels per million naive CD4 + cells or TREC levels per million total CD4 + cells in (F), because very few naive CD4 + cells express IFN-γ. Trend lines depict the differences in TREC levels among the four sorted populations in three patients. ( G ) CXCL8 production was determined following activation in vitro with PI (4 h, in the presence of BFA) in primary T-ALL samples ( n = 12); later stages (T-III/T-IV) are represented by gray diamonds.

Article Snippet: For CB, mononuclear cells were enriched in CD34 + cells (STEMCELL Technologies), and the CD34 − fraction (on occasion from pooled donors) was used for experiments.

Techniques: In Vivo, Irradiation, Derivative Assay, Expressing, Staining, Activation Assay, In Vitro

Journal: The Journal of Immunology Author Choice

Article Title: Adaptive from Innate: Human IFN-γ + CD4 + T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults

doi: 10.4049/jimmunol.1700551

Figure Lengend Snippet: CXCL8 production is imprinted in the thymus. CXCL8 production was determined following activation in vitro with PI (4 h, in the presence of BFA) in whole thymocytes versus peripheral blood from humanized mice (37 wk post–hematopoietic stem cell inoculation, n = 7) ( A ), sorted human thymocyte subsets ( n = 15, CD4 − CD8 − DN, CD4 + CD8 + double positive, CD4 − CD8 + SP CD8, CD4 + CD8 − SP CD4 + ) ( B ), and enriched T-lineage–committed DN thymocytes (defined as CD4 − CD8 − CD34 + CD7 + CD5 + CD1a + , n = 6) ( C ). Example and cumulative data are shown. Stimulation: BFA only in dark gray and PI+BFA in light gray. ( D ) Comparison of CXCL8 production between paired human SP CD4 + thymocytes and naive (CD31 + CD45RA + ) peripheral CD4 + T cells ( n = 10). * p < 0.05, ** p < 0.001.

Article Snippet: For CB, mononuclear cells were enriched in CD34 + cells (STEMCELL Technologies), and the CD34 − fraction (on occasion from pooled donors) was used for experiments.

Techniques: Activation Assay, In Vitro, Comparison

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 2. Expression and enzymatic activity of human r15-LOX (hr15-LOX) during induced erythroid maturation of K562 cells. A, detection of endog- enous hr15-LOX or GAPDH mRNA and protein in cytoplasmic extracts of K562 cells by RT-PCR and Western blot assays. B, hr15-LOX activity assays and HPLC analysis of the specific reaction product 15-HETE at days 0 and 8. Inset: the enantiomer composition of 15-HETE was analyzed by chiral phase HPLC. The profile indicates the preferential formation of the specific r15-LOX product 15(S)-HETE. C, non-induced K562 cells were transfected with control siRNAs (ctrl.) or siRNAs against hnRNP K and E1, as indicated. Cytoplasm was visualized by phalloidin-TRITC, nuclei by DAPI staining. hnRNP K, hnRNP E1, and hr15-LOX were detected by specific antibodies. The knockdown of hnRNP K and E1 was verified by a Western blot assay shown in Fig. 6A.

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Control, Staining, Knockdown

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 3. Expression of hr15-LOX mRNA translation regulators during induced erythroid maturation of K562 cells. A, cytoplasmic extracts of K562 cells from days 0 to 8 were resolved by SDS-PAGE. The expression of hnRNP E1, total hnRNP K, non-methylated hnRNP K, PRMT1, Lyn, c-Src, and GAPDH as a loading control was determined by specific antibodies in Western blot assays. B, the antibody non-RmetK recognizes specifically non-methylated hnRNP K in extracts from ES cells that do not express PRMT1 (ES/) (lane 2) and recombinant hnRNP K expressed in E. coli (lane 3), but not asymmetrically dimethylated hnRNP K in (ES/) cells (lane 1). A commercial monoclonal antibody against hnRNP K detects both, the methylated and non-methylated hnRNP K (lanes 1-3). C, 9 pmol of recombinant hnRNP K (lanes 2, 5, and 8) or the arginine substitution variant hnRNP K 5RG as a specificity control (lanes 3, 6, and 9) were incubated with [14C]S-adenosylmethionine and cytoplasmic K562 cell extract at day 0 (lanes 1–3) or day 8 (lanes 4–9) of induction in the absence (lanes 1–6) or presence (lanes 7–9) of 2.3 pmol of recombinant PRMT1, followed by SDS-PAGE and autoradiography. D, the level of mRNAs coding for hnRNP K, c-Src, and GAPDH was analyzed by RT-PCR. E, immunoprecipitation of total hnRNP K with the commercial monoclonal antibody from cytoplasmic extracts of K562 cells followed by SDS-PAGE and Western blot assays. The level of immunoprecipitated protein was detected with the hnRNP K antibody (hnRNP K), and the phosphorylated protein was detected with an anti-phosphotyrosine antibody (p-Tyr) and co-immunoprecipitated c-Src with the c-Src antibody (c-Src).

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Expressing, SDS Page, Methylation, Control, Western Blot, Recombinant, Variant Assay, Incubation, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 4. Expression of hr15-LOX and its translational regulators in human CD34 cells induced for terminalerythroidmaturation.A,benzidinestainingofhemoglobinduringerythroidmaturation.B,thelevel of endogenous -globin, hr15-LOX, and c-Src mRNAs in non-induced CD34 cells and at day 6 of the induction period was analyzed by RT-PCR. C–G, immunostaining of CD34 cells during induction. Nuclei were stained with DAPI. Erythroid maturation was monitored by staining of glycophorin A (GPA) and CD34 (except F; both the CD34 and the PRMT1 antibody were raised in rabbit). Four representative cells are shown in every panel. Enucleated cells are marked by an arrow. C, immunostaining of hr15-LOX. D, detection of hnRNP E1 with a specific antibody. E, a specific antibody was used to detect hnRNP K. F, immunostaining of PRMT1 and hnRNP K (instead of CD34). G, detection of c-Src by immunostaining.

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 5. hnRNP K directly binds to the c-Src mRNA 3-UTR, which mediates translational inhibition by blocking 80 S complex formation. A, immu- noprecipitated hnRNP K from cytoplasmic extract from non-induced cells (lane 2) and from cells at day 8 of induction (lane 5) was detected by Western blotting. For the control immunoprecipitation (ctrl.) an unrelated monoclonal antibody was used (lanes 3 and 6). Co-immunoprecipitated mRNAs were detected by RT-PCR as indicated. B, upper panel: schematic representation of the c-Src mRNA. Lower panel: 32P-labeled fragments of the c-Src mRNA 3-UTR, Src1 (nt 1–600, lanes 1–3), Src2 (nt 601–1200, lanes 4–6), or Src3 (nt 1201–2038, lanes 7–9) were incubated with 1.8, 3.6, and 7.2 pmol of recombinant hnRNP K. The 32P-labeled Src3 was incubated with 7.2 pmol of hnRNP K (lane 10) in the presence of 10- or 100-fold molar excess of unlabeled competitor RNAs Src1 (lanes 11 and 12), Src2 (lanes 13 and 14), Src3 (lanes 15 and 16), or DICE (lanes 17 and 18). C, cytoplasmic extract of non-induced K562 cells was used in cell-free translation reactions programmed with 32P-trace-labeled reporter mRNAs CAT, CAT-hDICE, CAT-Src3, or no RNA. Translation reactions were carried out in the presence of [35S]methionine. Translation products were resolved by SDS-PAGE and analyzed by autoradiography. The percentage of translated CAT protein is indicated below. 32P-trace-labeled mRNAs were extracted from the translation reactions at time point 0 and after 1 h, as indicated. The 32P-trace-labeled short CAT (sCAT) mRNA was used as an extraction control. The extracted mRNAs were separated on an agarose gel and analyzed by autoradiography. D, fractionation of translation initiation reactions containing cytoplasmic extract of non-induced K562 cells and 32P- labeled reporter mRNAs sORF-ctrl, sORF-Src3, or sORF-hDICE in the presence of cycloheximide on 5–25% sucrose gradients and analysis by scintillation counting. Positions of 80 S and RNP-containing fractions are indicated.

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Inhibition, Blocking Assay, Western Blot, Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Labeling, Incubation, Recombinant, SDS Page, Autoradiography, Extraction, Agarose Gel Electrophoresis, Fractionation

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 6. SiRNA-mediated knockdown of hnRNP K, but not hnRNP E1, causes de-repression of c-Src synthesis. A and B, non-induced K562 cells were transfected with control (ctrl.), hnRNP K, or hnRNP E1 siRNAs as indicated. A, detection of endogenous hnRNP K, hnRNP E1, GAPDH, or c-Src protein in cytoplasmic extracts of K562 cells at 0 and 48 h by Western blot assays. B, immunostaining of hnRNP K and E1 as in Fig. 2C. c-Src was detected with a specific antibody. Staining of the cytoplasm and nuclei are as in Fig. 2C.

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Knockdown, Transfection, Control, Western Blot, Immunostaining, Staining

Journal: Journal of Biological Chemistry

Article Title: mRNA Silencing in Human Erythroid Cell Maturation

doi: 10.1074/jbc.m710328200

Figure Lengend Snippet: FIGURE 7. Model of post-transcriptional regulation of gene expression in erythroid cell maturation. r15-LOX is a key enzyme in erythroid cell maturation. The synthesis of r15-LOX is restricted to mature reticulocytes, where the enzyme initiates the degradation of mitochondria as a prerequisite for erythrocyte formation (12). Left: in erythroblasts the translation of r15-LOX mRNA is silenced, because the formation of translation competent 80 S ribosomes on r15-LOX mRNA is inhibited by hnRNP K and E1, which bind to the 3-UTR DICE. This complex blocks the joining of the 60 S ribosomal subunit to the 40 S subunit at the AUG (16, 17). hnRNP K also binds to the c-Src mRNA 3-UTR element Src3 and inhibits the c-Src mRNA translation by blocking 80 S ribosome formation (this study). hnRNP K is quantitatively asymmetrically dimethylated on five arginine residues by PRMT1 (20). Erk-dependent phosphorylation of hnRNP K on Ser284 and Ser353 leads to its cytoplasmic accumulation (47). Right: in mature reticulocytes hnRNP K is released from the element Src3 in the c-Src mRNA 3-UTR, and the kinase is synthesized. Methylated hnRNP K is exchanged by the non-methylated form during erythroid maturation (this study) and non-methylated hnRNP K functions as a specific activator of c-Src (20, 21). The c-Src-dependent phosphorylation of Tyr458 in KH domain 3 of hnRNP K leads to the loss of DICE binding activity and consequently its role as an inhibitor of r15-LOX mRNA translation (19). r15-LOX mRNA translation is activated, and the newly synthesized enzyme catalyzes the dioxygenation of phospholipids in mitochondrial membranes (11–13).

Article Snippet: Antibodies—Antibodies were purchased from Santa Cruz Biotechnology (hnRNP K, Lyn, Fyn, p-Tyr, CD34, glycophorin A), Oncogene (v-Src), Abcam (GAPDH and PRMT1) and Sigma ( -tubulin) and used according to the manufacturer’s protocol.

Techniques: Gene Expression, Blocking Assay, Phospho-proteomics, Synthesized, Methylation, Binding Assay, Activity Assay