Journal: Leukemia

Article Title: Overexpression of wild-type or mutants forms of CEBPA alter normal human hematopoiesis

doi: 10.1038/leu.2012.38

Figure Lengend Snippet: Effect of expression of WT C/EBPα (dark grey bars), mutated C/EBPα on N-terminal (light grey) and on C-terminal (black) was evaluated on mouse hematopoietic progenitors by Colony Forming Units assay. 2×10 3 mouse hematopoietic progenitors (mLin − cells) after transduction were plated in M3434 medium; one week later number and type of CFUs were determinate. Serial replating was performed to evaluate secondary, tertiary, fourth or fifth CFUs respectively. Numbers of transduced CFU by Control (white bar), WT C/EBPα (dark grey), N-terminal (light grey) and C-terminal (black) are represented. The increase in colonies after serial replating observed could be explained by the presence of live immature cells in the methylcellulose, which were able after replating to mature and give rise to CFUs. Results are shown as mean ± standard deviation (SD), with significant differences of p ≤ 0.05, p ≤ 0.01 or p ≤ 0.001 are indicating by (*), (**) or (***) respectively.

Article Snippet: Infected Lin − cells were plated in triplicate in methylcellulose media (Methocult H4434 and Methocult M3434 for human and mouse respectively, StemCells Technologies, Vancouver, Canada) to assess Colony Forming Units (CFU).

Techniques: Expressing, Transduction, Control, Standard Deviation

Journal: Blood Advances

Article Title: Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice

doi: 10.1182/bloodadvances.2017013243

Figure Lengend Snippet: Hhex induces self-renewal of promyelocytes. (A) Wild-type LSK cells were retrovirally transduced with empty (MIG) or Hhex-encoding retroviruses, cultured in methylcellulose in the presence of IL-3, SCF, and EPO, and colony counts determined weekly. (B) Cumulative cell counts of methylcellulose cultures as in panel A. (C) Representative fields from week 4 cultures of MIG and MIG-Hhex–transduced LSK cells (original magnification ×400). (D) Cells harvested from methylcellulose cultures at the indicated round of plating were cytocentrifuged, stained with May-Grünwald-Giemsa, and the proportion of each cell type was determined by morphological examination. Data are representative of 3 separate experiments. (E) May-Grünwald-Giemsa–stained cytocentrifuge preparation of week 4 Hhex-transformed promyelocytes (original magnification ×400). (F) Fluorescence-activated cell sorter (FACS) plots of phenotypic markers (Mac-1, Kit, and Gr-1) and GFP expression in a representative Hhex-induced promyelocyte cell line. E, erythroblast; G, neutrophilic granulocyte; M, monocyte/macrophage; pro, promyelocyte.

Article Snippet: For colony assays, cells were suspended in mouse methylcellulose base media (R&D Systems) in the presence of the growth factors mentioned in the previous paragraph and colony numbers scored after 7 days.

Techniques: Transduction, Cell Culture, Staining, Transformation Assay, Fluorescence, Expressing

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Experimental layouts to determine the role of Nϕ in the A radioprotective and E radiomitigative effects of entolimod in BALB/c mice. Survival by Kaplan–Meier curves in mice treated with entolimod in the presence (rat IgG) or absence (α-Ly-6G) of Nϕ in the B radioprotective and F radiomitigative schemes. P -values were determined by Log-rank test. Measurement of total HPPs and granulocyte/macrophage (G/M) progenitors in BM by MethoCult in the C radioprotective ( n = 4–5 mice /group) and G radiomitigative ( n = 3–10 mice/group) schemes. Total HPPs were measured +1 h post-TBI for radioprotection and for radiomitigation, both total HPPs and G/M progenitors +3d post-treatment with vehicle or entolimod. D Absolute number of stem cell populations in BM by flow cytometry in the presence (rat IgG) and absence (α-Ly-6G) of Nϕ on day 8 post-TBI in the radioprotection scheme ( n = 3–5 mice/group). Stem cell populations were defined as follows: HSC (Lineage − Flt3 − c-kit + Sca-1 + ), MMP (Lineage − Flt3 + c-kit + Sca-1 + ), CLP (Lineage − Flt3 − IL-7R + c-kit + Sca-1 + ), Myeloid (Lineage − Flt3 − IL-7R − c-kit − Sca-1 − ), CMP (Lineage − Flt3 − IL-7R − CD34 + CD16/32 − ), MEP (Lineage − Flt3 − IL-7R − CD34 − CD16/32 − ), and GMP (Lineage − Flt3 − IL-7R − CD34 + CD16/32 + ). Error bars represent mean ± SEM; P -values were determined by Student’s t -test.

Article Snippet: We purchased mouse MethoCult GF and MethoCult without cytokines from STEMCELL Technologies, recombinant mouse G-CSF from Peprotech, rat IgG2a, κ (clone 2A3) and α-Ly-6G (clone 1A8) from BioXcell, Proteome Profiler Mouse XL Cytokine Array from R&D Systems, Duoset ELISA kits from R&D Systems, and recombinant mouse MMP-9 from BioLegend.

Techniques: Flow Cytometry

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: Tlr5 −/− bone marrow chimeric mice received an adoptive transfer of either Tlr5 WT/WT or Tlr5 −/− Nϕ (10 million /mouse) followed immediately by vehicle or entolimod 24 h post-TBI. A , B Survival was measured by Kaplan–Meier curves. P -values were determined by Log-rank test; n = 10–16 mice/group. C Measurement of total HPPs by MethoCult in BM on day 7 post-treatment using the same experimental setup as A . Error bars represent mean ± SEM; P -values were determined by Student’s t -test; n = 4–7 mice/group. In all instances, Nϕ purity was routinely >98%.

Article Snippet: We purchased mouse MethoCult GF and MethoCult without cytokines from STEMCELL Technologies, recombinant mouse G-CSF from Peprotech, rat IgG2a, κ (clone 2A3) and α-Ly-6G (clone 1A8) from BioXcell, Proteome Profiler Mouse XL Cytokine Array from R&D Systems, Duoset ELISA kits from R&D Systems, and recombinant mouse MMP-9 from BioLegend.

Techniques: Adoptive Transfer Assay

Journal: Cell Death Discovery

Article Title: Signaling through TLR5 mitigates lethal radiation damage by neutrophil-dependent release of MMP-9

doi: 10.1038/s41420-021-00642-6

Figure Lengend Snippet: A BALB/c mice ( n = 5/group) were given 5 Gy TBI followed 24 h later by treatment with vehicle, entolimod, or the indicated doses of recombinant pro-MMP-9 (rMMP-9). Measurement of total HPPs in BM by MethoCult was done on day 3 post-treatment. B Survival by Kaplan–Meier curve in BALB/c mice treated with vehicle, entolimod, or 10 μg/kg rMMP-9 24 h post 7.5 Gy TBI ( n = 9–10 mice /group). P -values were determined by the Log-rank test. C BALB/c mice ( n = 5/group) were given 5 Gy TBI followed 6 h later by rat IgG or α-Ly-6G. Twenty-four hours later mice were treated with vehicle, entolimod, rMMP-9 (10 μg/kg), or rMMP-9 plus entolimod. Measurement of total HPPs in BM by MethoCult was done on day 3 post-treatment. For A and C error bars represent mean ± SEM; P -values were determined by Student’s t -test.

Article Snippet: We purchased mouse MethoCult GF and MethoCult without cytokines from STEMCELL Technologies, recombinant mouse G-CSF from Peprotech, rat IgG2a, κ (clone 2A3) and α-Ly-6G (clone 1A8) from BioXcell, Proteome Profiler Mouse XL Cytokine Array from R&D Systems, Duoset ELISA kits from R&D Systems, and recombinant mouse MMP-9 from BioLegend.

Techniques: Recombinant