Journal: Nature

Article Title: Pulmonary Macrophage Transplantation Therapy

doi: 10.1038/nature13807

Figure Lengend Snippet: ( a-b ) Photomicrographs of WT BMDMs prior to transplantation phase-contrast ( a ) or Diff-Quick staining ( b ) (Representative of n=7 BMDM preparations). Scale bar, 20 μm. ( c ) Flow cytometry evaluation of cell-surface phenotypic markerson WT BMDMs before PMT. ( d ) Photographs of methylcellulose cultures of Lin − cells (5,000/dish) from bone marrow (left) and BMDMs (50,000/dish) prepared as described in the Methods (right) and typical colonies (below) (representative n=3 per condition). ( e ) Colony counts of BFU-E, CFU-GEMM and CFU-GM showing BMDMs contained <0.005% CFU-GM and no BFU-E or CFU-GEMM progenitors, corresponding to 93 CFU-GM per dose of BMDMs administered (n=3 determinations per condition). ( f-g ) Evaluation of surfactant clearance capacity. Representative photomicrographs of BMDMs from WT (left) or KO (right) were examined before (top) or immediately after incubation with surfactant for 24 hours (middle), or after exposure, removal of extracellular surfactant and culture for 24 hours in the absence of surfactant (lower) after oil-red-O staining (Representative of n=3 per condition). Scale bar, 20 μm. ( g ) Measurement of surfactant clearance by BMDMs after exposure as just described (Panel f ) and quantified using a visual grading scale (the oil-red-O staining index) to measure the degree of staining. Bars represent the mean ± SEM (n=3/condition) of oil-red-O staining score for 10 high-power fields for each group. Not detected (ND).***P<0.001.

Article Snippet: Briefly, fresh Lin − bone marrow cells or BMDMs after induced differentiation into macrophages for five days were seeded into standard mouse methylcellulose media supplemented with insulin, transferrin, SCF, IL-3, IL-6, and erythropoietin (HSC007, R&D Systems, Minneapolis, MN).

Techniques: Transplantation Assay, Diff-Quik, Staining, Flow Cytometry, Incubation

Journal: Acta biomaterialia

Article Title: Lipopolymer/siRNA Complexes Engineered for Optimal Molecular and Functional Response with Chemotherapy in FLT3-Mutated Acute Myeloid Leukemia.

doi: 10.1016/j.actbio.2024.08.053

Figure Lengend Snippet: Figure 4. Molecular response to treatment with lipopolymer/siRNA complexes in MV4-11 cells. (A) FLT3 gene levels assessed over time by RT-qPCR. (B) Histogram overlays demonstrating a negative shift in the population due to loss of anti-FLT3 antibody fluorescence and recovery with time. FLT3 protein levels over time depicted as (C) mean fluorescence intensity (MFI) (normalized to that of untreated cells) of the anti-FLT3 antibody, and (D) percentage of FLT3 negative cell population. (E) Colony formation ability evaluated by counting number of colonies at days 7 and 14 after methylcellulose seeding of treated cells. **p<0.005, *p<0.05; Student’s two-tailed t-test assuming unequal variance. UTC – Untreated Control.

Article Snippet: 200 cells were seeded in 400 μL of human methylcellulose base media (R&D Systems, Minneapolis, MN) supplemented with 10% RPMI and transferred to a 24-well plate.

Techniques: Quantitative RT-PCR, Fluorescence, Two Tailed Test, Control

Journal: Nucleic Acids Research

Article Title: IRF2BP2 counteracts the ATF7/JDP2 AP-1 heterodimer to prevent inflammatory overactivation in acute myeloid leukemia (AML) cells

doi: 10.1093/nar/gkae437

Figure Lengend Snippet: IRF2BP2 depletion influences differentiation from mouse bone marrow stem and progenitor cells. ( A ) Western blot of components of the IRF2BP2 complex in cells isolated from mouse bone marrow. NUC = nucleoplasm, CHR = chromatin. ( B ) Representative bright field photography of colonies (here CFU-GM) obtained upon colony formation assays of myeloid progenitor cells with and without IRF2BP2 KD in methylcellulose medium. Additional examples are shown in . Scale = 500 μm. ( C ) Quantification of the area of colonies upon differentiation in methylcellulose. For each condition, 15 colonies were measured from two biological replicates. Significance was evaluated via a two-tailed unpaired Student's t -test. ( D ) Representative bright field microscopy photography of dendritic cell-shaped cells (arrows), seen in the IRF2BP2 KD cells compared to the control cells. Scale = 50 μm. ( E ) RT-qPCR analysis of collected cells after colony formation assays. Data represents the mean of three biological replicates. Significance was evaluated via a two-tailed paired Student's t -test. ( F ) FACS quantification of surface makers after 10 days of differentiation of Lin-negative bone marrow cells in liquid culture. Data represent the mean ± s.d. of at least three biological replicates. Significance was evaluated via a two-tailed unpaired Student's t -test. Example histograms are shown in . ( G ) GSEA of TCGA and our own RNA-Seq data regarding pathways involved in myeloid leukocyte migration and stem cell maintenance. n.s. = no significant; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The cells were seeded in duplicates at 5 × 10 3 in 1 ml of mouse methylcellulose complete media without Epo (HSC008; R&D Systems) containing mIL-3, mIL-6 and mSCF (concentrations as described above), on 35 mm dishes and cultivated at 37°C and 5% CO 2 .

Techniques: Western Blot, Isolation, Two Tailed Test, Microscopy, Control, Quantitative RT-PCR, RNA Sequencing, Migration