Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: E10.5 (left panels) and E12.5 (right panels) placentas were dissociated to a single cell suspension and analyzed. Prom1+Sca1+CD34+ cells are displayed for (A) FCS/SSC, CD49f and (B) CD31, CD105, VE-Cadherin, Tie2, CD41 and cKit expression. Red boxes highlight the phenotype Prom1+Sca1+CD45− cells.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: Placentas from (A) E10.5, (B) E11.5 and (C) E12.5 were stained with antibodies against CD133 (Prom1, green). The left panels show composite low magnification pictures of transversal sections of placentas. The region of the labyrinth that contains Prom1+ cells is highlighted (white circles).

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Staining

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) Hemogenic PS34 cells from E10.5 and E12.5 placentas were fractioned into CD45−cKit−, CD45−cKit+ and CD45+cKit+ populations. HSPCs and mature blood cells (MBC) were also sorted for comparison.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques:

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E10.5 AGM+VU regions were isolated, dissociated to a single cell suspension and analyzed by flow cytometry. PS34CD45− and PS34CD45+ cells are gated and displayed for the expression of the 23GFP Runx1 reporter. Percentages are shown as mean ± SD, n=3.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Developmental cell

Article Title: Hematopoietic Reprogramming in vitro Informs in vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells

doi: 10.1016/j.devcel.2016.02.011

Figure Lengend Snippet: (A) E12.5 PS34CD45− cells were co-cultured on OP-9 in the presence of cytokines (SCF, Fltl3L, IL-3, IL-6 and TPO) for 4 days. FACS analysis shows the generation of CD45+CD34+cKit+ cells that were tested for clonogenic activity.

Article Snippet: Ao, Aorta. ( D ) Fluidigm qRT-PCR analysis of Prom1 , Ly6a and CD45 in E10.5 AGM+VU endothelial (EC: CD144+(VE-Cadherin)Ter119−CD45−CD41−23GFP−), hemogenic endothelium (HE: CD144+Ter119−CD45−CD41−23GFP+) and HSPCs (CD144+Ter119−CD45−CD41+23GFP+) (mean ± SD, n=3).

Techniques: Cell Culture, Activity Assay

Journal: Biomolecules

Article Title: Clinical Significance and Immune Infiltration Analyses of the Cuproptosis-Related Human Copper Proteome in Gastric Cancer

doi: 10.3390/biom12101459

Figure Lengend Snippet: Immune infiltration analyses. ( A – C ) Detection of infiltrated macrophages, CD8+ T cells and CD4+ T cells in 4 responders/CBP_low and 4 non-responder/CBP_high to immunotherapy in the PKUCH cohort. ( D – F ) The M1/M2 ratio, CD8+ T cell proportion and CD4+ T cell proportion in 4 responders/CBP_low and 4 non-responders/CBP_high to immunotherapy. ( G – I ) Kaplan–Meier curves for the OS of GC patients in different groups classified by the median of CD68, CD8 and CD4 levels from a PKUCH cohort.

Article Snippet: FFPE tissue slides were first deparaffinized in a BOND RX system (Leica Biosystems) and then incubated sequentially with primary antibodies targeting CD4 (1:100: Abcam, ab133616), CD8 (1:200, Abcam, ab178089), CD163 (1:500, Abcam, ab182422), CD68 (1:1000, Abcam, ab213363), PD-1 (1:200, CST, D4W2J, 86163S), PD-L1 (1:400, CST, E1L3N, 13684S) and CD20 (1:1, Dako, L26, IR604).

Techniques:

Journal:

Article Title: Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation

doi: 10.1111/j.1399-3089.2008.00482.x

Figure Lengend Snippet: Immunohistochemical staining of porcine islet grafts retrieved 24 hours after intraportal xenotransplantation from diabetic monkeys treated with LMW-DS or heparin. The figure shows representative expression of insulin and of CD41 (platelets), CD68 (macrophages), and CD3 (T-cells) in the grafts. A summary of all transplanted monkeys is presented in Table 1. Magnification x200.

Article Snippet: Immunohistochemical staining was carried out using guinea pig anti-insulin (DAKO, Carpenteria, CA, USA), mouse anti-human neutrophil elastase (DAKO), mouse anti-human CD68 (DAKO), mouse anti-human MAC387 (Abcam, Cambridge, UK), mouse anti-human CD56 (Monosan, Stockholm, Sweden), rabbit anti-human CD3 (DAKO), mouse anti-human CD20 (DAKO), rabbit anti-human IgG and IgM (DAKO), mouse anti-human CD41 (DAKO), mouse anti-human C3c (QUIDEL, San Diego, CA, USA), or goat anti-human C9 (Serotec Ltd Scandinavia, Oslo, Norway).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal:

Article Title: Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation

doi: 10.1111/j.1399-3089.2008.00482.x

Figure Lengend Snippet: Summary of the immunohistochemical staining (as depicted in ) of the islet grafts in recipient monkeys receiving LMW-DS or heparin

Article Snippet: Immunohistochemical staining was carried out using guinea pig anti-insulin (DAKO, Carpenteria, CA, USA), mouse anti-human neutrophil elastase (DAKO), mouse anti-human CD68 (DAKO), mouse anti-human MAC387 (Abcam, Cambridge, UK), mouse anti-human CD56 (Monosan, Stockholm, Sweden), rabbit anti-human CD3 (DAKO), mouse anti-human CD20 (DAKO), rabbit anti-human IgG and IgM (DAKO), mouse anti-human CD41 (DAKO), mouse anti-human C3c (QUIDEL, San Diego, CA, USA), or goat anti-human C9 (Serotec Ltd Scandinavia, Oslo, Norway).

Techniques: Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

doi: 10.18632/oncotarget.15226

Figure Lengend Snippet: A. Expression levels of miR-494-3p in CB CD34+ cells were evaluated 24, 48 and 96 hours after the last nucleofection by means of qRT-PCR. Data are reported as RQ mean ± S.E.M of 5 independent experiments. Results were normalized to mimic-NegCTR sample and U6 was selected as endogenous control. B. Subpanels i and ii represent statistical analysis of flow cytometry evaluation of CD34 and CD38 protein expression in CB CD34+ cells cultured in multilineage conditions in the presence of HS at 96 hours upon mimic nucleofection (n=2). Subpanel iii shows the flow cytometry analysis of a representative experiment. Subpanel iv represents the absolute numbers of cells belonging to the three different populations: CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+. Absolute cell numbers were calculated, according to the percentage of cells for each population, starting from the average total cell number in each sample. C-D. Flow cytometry analysis of expression of monocytic (CD14, CD163), granulocytic (CD15, CD66b, MPO), megakaryocytic (CD41) and erythroid (GPA) differentiation markers in CB CD34+ cells overexpressing miR-494-3p maintained in multilineage conditions in the presence of HS (C) or the serum substitute BIT 9500 (D) at day 11 of cell culture. (n=3) E. Results of the statistical analysis of methylcellulose clonogenic assay of CB CD34+ cells overexpressing miR-494-3p. Cells were plated 24 hours after mimic nucleofection and colonies were scored at day 14 (n=3). Results are reported as mean ± S.E.M. *, p≤0.05 Abbreviations: CFU, colony-forming unit; BFU, burst-forming unit; E, erythroid; GM, granulo-monocyte; G, granulocyte; M, monocyte; GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte.

Article Snippet: The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb; allophycocyanin (APC)-conjugated mouse anti-human CD34 MoAb; FITC-conjugated mouse anti-human CD66b MoAb; FITC-conjugated mouse anti-human CD15 MoAb; phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb; APC-conjugated mouse anti-human CD163 MoAb; PE-conjugated mouse anti-human CD42b MoAb (all from Miltenyi Biotech); FITC-conjugated mouse anti-human CD41 MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia; http://www.dako.com ) and FITC-conjugated mouse anti-human MPO MoAb (from BD Biosciences; San Jose, CA USA).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Cell Culture, Clonogenic Assay

Journal: Oncotarget

Article Title: miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

doi: 10.18632/oncotarget.15226

Figure Lengend Snippet: A-B. Bar-graphs represent the statistical analysis of the flow cytometry evaluation of CD41 (A) and CD42b (B) cell markers in CB CD34+ cells overexpressing miR-494-3p. Marker expression was assessed at days 3, 5, 8, 10 and 12 of serum-free MK unilineage cell culture (n=3). C. Flow cytometry analysis of co-expression of CD41 and CD42b surface antigens at days 8 and 10 of MK unilineage culture after mimic nucleofection in CB CD34+ cells (n=3). D. Representative morfological analysis of mimic-NegCTR (i-iii) and mimic-494 (ii-iv) samples after May-Grünwald-Giemsa staining at days 8 and 10 of MK unilineage culture. Magnification, x400. Scale bar: 100 μm. E. Subpanel i represents the statistical analysis of flow cytometry analysis of co-expression of CD34 and CD41 surface antigens at days 4 and 6 post mimic electroporation in serum free multilineage culture (n=3). Subpanel ii shows corresponding dot plot graphs of a representative experiment. F. Results of the statistical analysis of collagen-based clonogenic assay of CB CD34+ cells overexpressing miR-494-3p. Cells were seeded in semisolid culture medium 24 hours after the last nucleofection and colonies were scored after 11 days (n=3). Results are reported as mean ± S.E.M. **, p≤0.01; *, p≤0.05 Abbreviations: CFU, colony-forming unit; MK, megakaryocyte; MIX, mixed; nonMK, other than megakaryocyte.

Article Snippet: The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb; allophycocyanin (APC)-conjugated mouse anti-human CD34 MoAb; FITC-conjugated mouse anti-human CD66b MoAb; FITC-conjugated mouse anti-human CD15 MoAb; phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb; APC-conjugated mouse anti-human CD163 MoAb; PE-conjugated mouse anti-human CD42b MoAb (all from Miltenyi Biotech); FITC-conjugated mouse anti-human CD41 MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia; http://www.dako.com ) and FITC-conjugated mouse anti-human MPO MoAb (from BD Biosciences; San Jose, CA USA).

Techniques: Flow Cytometry, Marker, Expressing, Cell Culture, Staining, Electroporation, Clonogenic Assay

Journal: Oncotarget

Article Title: miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

doi: 10.18632/oncotarget.15226

Figure Lengend Snippet: A-B. Results of the statistical analysis of the percentages of CD41+ and CD42b+ cells evaluated by means of flow cytometry upon SOCS6 silencing in CB CD34+ cells. Marker expression was assessed at days 3, 5, 8, 10 and 12 in cells cultured in serum-free MK unilineage conditions (n=4). C. Flow cytometry analysis of cells co-expressing CD41 and CD42b surface antigens at days 8 and 10 of MK unilineage culture (i) (n=4). Sub-panel ii displays flow cytometry graphs of a representative experiment. D. Morphological analysis of siNeg CTR (i-iii) and siSOCS6 (ii-iv) samples after May-Grünwald-Giemsa staining at days 8 and 10 of MK unilineage culture in a representative experiment. Magnification, x400. Scale bar: 100 μm. E. Results of flow cytometry analysis of cells expressing both CD34 and CD41 surface antigens at days 3 and 5 post electroporation in serum free multilineage culture (n=4). F. Results of the statistical analysis of collagen-based clonogenic assay of CB CD34+ cells upon SOCS6 silencing. Cells were seeded in semisolid culture medium 24 hours after the last nucleofection and colonies were scored after 11 days (n=3). G. miR-494-3p and SOCS6 expression kinetic during MK differentiation. Gene and miRNA expression levels were normalized to cells cultured in the presence of HS for 24 hours after purification (HS 24 sample). 18s rRNA and U6 were used as endogenous controls for gene and miRNA expression evaluation respectively (n=4). Results are reported as mean ± S.E.M. **, p≤0.01; *, p≤0.05 Abbreviations: CFU, colony-forming unit; MK, megakaryocyte; MIX, mixed, nonMK, other than megakaryocyte.

Article Snippet: The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb; allophycocyanin (APC)-conjugated mouse anti-human CD34 MoAb; FITC-conjugated mouse anti-human CD66b MoAb; FITC-conjugated mouse anti-human CD15 MoAb; phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb; APC-conjugated mouse anti-human CD163 MoAb; PE-conjugated mouse anti-human CD42b MoAb (all from Miltenyi Biotech); FITC-conjugated mouse anti-human CD41 MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia; http://www.dako.com ) and FITC-conjugated mouse anti-human MPO MoAb (from BD Biosciences; San Jose, CA USA).

Techniques: Flow Cytometry, Marker, Expressing, Cell Culture, Staining, Electroporation, Clonogenic Assay, Purification

Journal: Oncotarget

Article Title: miR-494-3p overexpression promotes megakaryocytopoiesis in primary myelofibrosis hematopoietic stem/progenitor cells by targeting SOCS6

doi: 10.18632/oncotarget.15226

Figure Lengend Snippet: A. Scatter plot representing the correlation between miR-494-3p and SOCS6 expression levels according to the microarray analysis in our initial dataset of 42 PMF samples and 31 healthy donors. The Spearman rank correlation coefficient (r) was used to measure and identify the degree of linear dependence between the ranked variables (r = −0.57, p = 1.12 × 10 −7 ). Green circles = PMF CD34+ cells, Red triangles = PB CD34+ cells, Blue squares = BM CD34+ cells. Abbreviations: PMF, primary myelofibrosis; PB, peripheral blood; BM, bone marrow; CTR, control. B. Western blot analysis of SOCS6 protein levels in PMF CD34+ cells compared to CB CD34+ cells. β-actin was included as loading control. C-D. Expression levels of miR-494-3p, SOCS6, and PTEN in PMF CD34+ cells after miRNA inhibition evaluated by means of qRT-PCR. Data are reported as RQ mean ± S.E.M (n=3). E. Immunoblots representing SOCS6 protein levels in whole cell lysates from PMF CD34+ cells after miR-494-3p inhibition. SOCS6 protein levels were evaluated 24, 48 and 96 hours after miR-494-3p inhibitors nucleofection and were compared with control samples electroporated with Negative Control inhibitors (NegCTR). β-actin was included as loading control while PTEN was selected as positive control. F-G. Results of flow cytometry analysis of the percentages of CD41+ (F) and CD42b+ (G) cells evaluated at days 3, 5, 8, 10 and 12 in serum-free MK unilineage conditions (n=3). H. Morfological analysis of Neg CTR Inhib (i) and miR-494 Inhib (ii) samples after MGG staining at day 9 of MK unilineage culture in a representative experiment. Magnification, x400. Scale bar: 100 μm. I. Results of the statistical analysis of collagen-based clonogenic assay of PMF CD34+ cells upon miR-494-3p inhibition. Cells were seeded in semisolid culture medium 24 hours after the last nucleofection and colonies were scored after 11 days (n=3). Results are reported as mean ± S.E.M. *, p≤0.05. Abbreviations: CFU, colony-forming unit; MK, megakaryocyte; MIX, mixed; nonMK, other than megakaryocyte.

Article Snippet: The following monoclonal antibodies (MoAbs) were used for flow cytometry analysis: fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 MoAb; allophycocyanin (APC)-conjugated mouse anti-human CD34 MoAb; FITC-conjugated mouse anti-human CD66b MoAb; FITC-conjugated mouse anti-human CD15 MoAb; phycoerythrin (PE)-conjugated mouse anti-human CD14 MoAb; APC-conjugated mouse anti-human CD163 MoAb; PE-conjugated mouse anti-human CD42b MoAb (all from Miltenyi Biotech); FITC-conjugated mouse anti-human CD41 MoAb, and PE-conjugated mouse anti-human GPA MoAb (all from Dako; Milano, Italia; http://www.dako.com ) and FITC-conjugated mouse anti-human MPO MoAb (from BD Biosciences; San Jose, CA USA).

Techniques: Expressing, Microarray, Western Blot, Inhibition, Quantitative RT-PCR, Negative Control, Positive Control, Flow Cytometry, Staining, Clonogenic Assay