Journal: Nature Communications

Article Title: Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment

doi: 10.1038/s41467-020-16911-4

Figure Lengend Snippet: a Schematic illustration of competitive bone marrow transplantation. b Chimerism studies in indicated tissues and bone marrow LSK cells. Representative flow cytometric plots illustrate the percentages of wild type competitive BMMCs (CD45.1 + ) and the control or mDia2 fl/fl Vav-Cre BMMCs (CD45.2 + ) before and 1.5-month after transplantation. c Quantitative analyses of the percentage of donor cells in b . Gran: Gr1+ Mac1+ granulocytes; MO: Gr1-Mac1+ monocytes; B: B220+ B cells; T: CD3e+ T cells. d Peripheral blood chimerism analyses at the indicated time points. b – d n = 5 mice per group. e mDia2 fl/fl or mDia2 fl/fl Vav-Cre bone marrow lineage negative (CD45.2+) HSPCs (2 × 10 7 cells) were mixed with equal wild-type CD45.1+ competitive lineage negative cells and transplanted into lethally irradiated wild type mice. Bone marrow chimerism studies were performed at 12 h post-transplantation. Representative data are shown and similar results were repeated twice. f Transwell migration assay of HSPCs in the presence or absence of SDF-1α. n = 2 mice in no serum group, n = 4 mice in SDF1a group. g Transwell migration assay of indicated lineage-negative cells in the presence of mouse endothelial cells (EC) with or without SDF-1α. n = 2 mice per group. h In vivo imaging of fluorescently labeled lineage negative donor cells from the femur of wild-type mice 18 h after competitive transplantation with an equal number (9 × 10 5 ) of non-fluorescently labeled CD45.1+ cells. Red, CD31+ endothelial cells. Green, CD45.2+ donor cells. Dashed white lines outline blood vessels. Repeated twice with similar results. i Positions of the indicated donor cells relative to the outlined CD31+ lining sinusoids or arteries were quantified in five fields. N = 39 in mDia2 fl/fl group. N = 18 in mDia2 fl/fl Vav-Cre group. j Representative flow cytometric plots showing percentage of c-Kit+ HSCPs among lineage negative cells after intra-peritoneal injection of G-CSF. k Colony forming assay of peripheral blood in j ( n = 4 mice per group). Colony-derived cells were quantified on the right ( n = 3 mice per group). Error bars represent the SEM of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Two-tailed unpaired Student’s t test was used to generate the p values.

Article Snippet: Lineage-negative HSPCs were isolated from the tibia and femoral marrow compartments by depletion of lineage-positive cells using a Lineage Cell Depletion Kit (Catalog # 559971 BD Pharmingen) according to the manufacture’s protocol.

Techniques: Transplantation Assay, Irradiation, Transwell Migration Assay, In Vivo Imaging, Labeling, Injection, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment

doi: 10.1038/s41467-020-16911-4

Figure Lengend Snippet: a Quantitative analysis of the flow cytometry detected surface expression levels of beta2 integrins in c-Kit+ HSPCs from mDia2 fl/fl or mDia2 fl/fl Vav-Cre (cKO) mice transduced with indicated genes. N = 3 mice in each group, except mDia2 ΔDAD and mDia2 GBD/DID groups. b Restoration of the mRNA expression of beta2 integrins and indicated SRF downstream genes by overexpression of SRF in c-Kit+ cells from mDia2 fl/fl Vav-Cre (cKO) mice. The experiments were performed in triplicate. N = 3 mice in each group. c Viral transduced c-kit+ HSPC from mDia2 fl/fl or mDia2 fl/fl Vav-Cre (cKO) mice were applied for in vitro adhesion assay with fibronectin precoated coverslip for 1 h at 37 °C. Adherent cells were randomly scored from N = 7 fields in mDia2 fl/fl +Vector, N = 9 fields in cKO+Vector, N = 8 fields in cKO+SRFΔ5, and N = 6 fields in cKO+SRF-FL. Results are expressed as the adhesion ability relative to control cells transduced with vector. d Schematic diagram showing bone marrow transplantation experiments in e – g . e Chimerism studies of peripheral blood mononuclear cells derived from c-Kit+ cells that were transduced with the indicated constructs after competitive transplantation with CD45.1+ cells at indicated timepoints. f Quantification of multi-lineage reconstitutions in the peripheral blood from e at 3-month. g LSK and long-term HSC engraftment analyzed in bone marrow from 6-month-old transplanted mice shown in e . h Kaplan–Meier survival curve of indicated groups of mice after secondary transplantation illustrated in d . Error bars represent the SEM of the mean. p values compared to cKO+Vec group from g – j are presented on the right table. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Two-tailed unpaired Student’s t test was used to generate the p values. Significance for survival analyses was calculated by log-rank (Mantel–Cox) test.

Article Snippet: Lineage-negative HSPCs were isolated from the tibia and femoral marrow compartments by depletion of lineage-positive cells using a Lineage Cell Depletion Kit (Catalog # 559971 BD Pharmingen) according to the manufacture’s protocol.

Techniques: Flow Cytometry, Expressing, Transduction, Over Expression, In Vitro, Cell Adhesion Assay, Plasmid Preparation, Transplantation Assay, Derivative Assay, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Diaphanous-related formin mDia2 regulates beta2 integrins to control hematopoietic stem and progenitor cell engraftment

doi: 10.1038/s41467-020-16911-4

Figure Lengend Snippet: a Experimental outline for the measurement of sgRNA genome editing activities by a luciferase reporter assay and subsequent editing specifically in hematopoietic cells from Cas9 EGFP transgenic mice through bone marrow transplantation. b Methodological validation of luciferase-based genome editing in Cas9 expressing 293T cells. Representative data performed in triplicate were shown. The experiments were repeated twice with similar results. c Quantitative analyses of the transduction efficiencies of the indicated sgRNAs in vivo in the hematopoietic cells by flow cytometry one month after transplantation. n = 2 mice in each group. d Evaluation of genome editing efficacies of sgRNAs in peripheral blood Gr1+ granulocytes. n = 3 mice per group except n = 2 mice in CD45.1 as negative control. e Same as d except LSK cells in the bone marrow were analyzed. n = 2 mice per group except n = 3 in sgItgb2 group. f Chimerism analysis in the peripheral blood of the recipient mice following secondary competitive bone marrow transplantation with 2 × 10 6 BMMC donor cells transduced with the indicated sgRNAs and equal number of CD45.1+ wild type BMMC competitors. n = 3 mice per group except n = 4 in sgLuc2P group. g Kaplan–Meier survival curve of indicated mice after secondary transplantation with 3.5 × 10 6 BMMCs from primary transplant recipients from c . n = 10 mice per group except n = 9 in sgItgb2 group. h c-Kit+ HSPCs transduced as indicated were applied for competitive transplantation as described in Fig. . Chimerism studies in the peripheral blood at 4 months after transplantation were shown on the left panel ( n = 4 mice in each group), and the long-term HSC engraftments in the bone marrow were on the right panel ( n = 6 mice per group except n = 5 in cKO+Vec group). i Chimerism analyses of multi-lineage reconstitutions in peripheral blood mononuclear cells derived from indicated transduced c-Kit+ cells after competitive transplantation with CD45.1+ cells. n = 2 mice in each group. Error bars represent the SEM of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Two-tailed unpaired Student’s t test was used to generate the p values. Significance for survival analyses was calculated by log-rank (Mantel–Cox) test.

Article Snippet: Lineage-negative HSPCs were isolated from the tibia and femoral marrow compartments by depletion of lineage-positive cells using a Lineage Cell Depletion Kit (Catalog # 559971 BD Pharmingen) according to the manufacture’s protocol.

Techniques: Luciferase, Reporter Assay, Transgenic Assay, Transplantation Assay, Expressing, Transduction, In Vivo, Flow Cytometry, Negative Control, Derivative Assay, Two Tailed Test