Journal: Cell Communication and Signaling : CCS

Article Title: M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

doi: 10.1186/s12964-018-0300-8

Figure Lengend Snippet: M2 macrophages promote myofibroblast differentiation of LR-MSCs through the Wnt/β-catenin signaling pathway. a RAW 264.7 cells were treated with LPS (10 ng/ml) or IL-4 (10 ng/ml) for 24 h to induce M1 and M2 macrophage differentiation, respectively. The mRNA expression levels of inducible nitric oxide synthase (iNOS, M1 macrophage marker), arginase (Arg-1, M2 macrophage marker), and Wnt7a in differentiated macrophage subtypes were determined by q-PCR. Results are expressed as means ± SD ( n = 5; * p < 0.05 vs. M1 macrophage). b Wnt7a levels in the culture supernatant of differentiated macrophage subtypes were determined by ELISA. Data were expressed as means ± SD ( n = 5; * p < 0.05 vs. control). c LR-MSC lysates were subjected to coimmunoprecipitation (Co-IP) with anti-Frizzled-1 antibody, and the blot was probed with anti-Wnt7a antibody. Moreover, blots were re-probed with anti-Frizzled-1 antibody to confirm equal protein loading. Co-IP with mouse IgG served as a negative control. The presence of Wnt7a in the cell lysate was detected by western blotting, serving as a positive control. d The ratios of Wnt7a/Frizzled-1 were determined by densitometry and were expressed as means ± SD ( n = 3; * p < 0.05 vs. control). e The nuclear translocation of β-catenin was evaluated by measuring protein levels in the cytosolic and nuclear extracts. Histone H3 and GAPDH were used as loading controls for nuclear and cytoplasmic proteins, respectively, and also used as a control for the purity of the preparation. f M2 macrophages were transfected with control or Wnt7a siRNA and then cocultured with LR-MSCs in a transwell system. The expression of α-smooth muscle actin (α-SMA) and collagen I in LR-MSCs was measured by western blotting. g and h Purified primary LR-MSCs were cocultured with M2 macrophages or control medium in a transwell system. In some of the cocultured LR-MSCs as indicated 1 μM salinomycin (a specific Wnt/FZD/LRP5 complex inhibitor) or the solvent DMSO was added to the medium of the cocultured LR-MSCs to block Wnt/β-catenin signaling. Expression of α-smooth muscle actin (α-SMA) and collagen I on LR-MSCs was measured by immunofluorescence assay ( g ) and western blotting ( h )

Article Snippet: Commercially available siRNA for the mouse Wnt7a (sc-41,115) gene and nonspecific control siRNA (sc-37,007) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Marker, Enzyme-linked Immunosorbent Assay, Control, Co-Immunoprecipitation Assay, Negative Control, Western Blot, Positive Control, Translocation Assay, Transfection, Purification, Solvent, Blocking Assay, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

doi: 10.1186/s12964-018-0300-8

Figure Lengend Snippet: M2 macrophages infiltration predominates in human IPF lung tissues. a The expression of iNOS (M1 macrophage marker) and CD206 (M2 macrophage marker) in IPF fibroblastic focus were examined by immunohistochemistry. Representative images are shown ( n = 7). b The expression of collagen I, β-catenin, Wnt7a, and α-smooth muscle actin (α-SMA) in human IPF lung tissues was measured by western blotting. The expression levels were quantified with ImageJ (lower panels; n = 3). GAPDH was used as a loading control. Results are normalized to the expression of each individual protein in the control which is given a value of 1 and are expressed as means ± SD (* p < 0.05 vs. Control). c Expression of Wnt7a protein in CD206 + M2 macrophages was measured by immunofluorescence assay. Arrow indicates individual cells positive for both CD206 (green) and Wnt7a (red). Representative images are shown

Article Snippet: Commercially available siRNA for the mouse Wnt7a (sc-41,115) gene and nonspecific control siRNA (sc-37,007) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Marker, Immunohistochemistry, Western Blot, Control, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

doi: 10.1186/s12964-018-0300-8

Figure Lengend Snippet: Schematic representation for hypothetical M2 macrophage-induced myofibroblast differentiation of LR-MSCs. Bleomycin induces severe local inflammation. The recruited M2 macrophages release high levels of Wnt7a, which promotes myofibroblast differentiation of LR-MSCs and pulmonary fibrosis through activating the Wnt/β-catenin signaling pathway

Article Snippet: Commercially available siRNA for the mouse Wnt7a (sc-41,115) gene and nonspecific control siRNA (sc-37,007) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Subnuclear cyclin D3 compartments and the coordinated regulation of proliferation and immunoglobulin variable gene repression

doi: 10.1084/jem.20120800

Figure Lengend Snippet: Alteration of PI3K signaling does not affect B cell progenitor proliferation. (A) B cell progenitors from WT and Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. Cells were then fixed and analyzed for DNA content and the percentage of cells in G 1 versus SG 2 M phase of cell cycle are provided. Data are representative of three independent experiments. (B) B cell progenitors from Bim −/− bone marrow were cultured in the presence of IL-7 with or without LY294002 for 24 h. TCLs were prepared in NP-40 lysis buffer, resolved by SDS-PAGE, and membranes probed for cyclin D3 and Actin. Relative molecular mass (kD) is as indicated. Data are representative of three independent experiments. (C) Flow cytometry for expression of B220, CD43, and IgM by bone marrow cells from p85α +/− and p85α −/− mice. Numbers in contour plots indicate the percentage of cells in each gate. Representative data, n = 8. (D) Relative number of cells in each population determined using total number of bone marrow cells after red blood cell lysis and percentages from flow cytometry. Each point represents a single mouse and bars represent the mean for each population. (E) The corresponding populations from C were analyzed for DNA content. Numbers in histogram plots indicate the percentage of cells in G 1 and SG 2 M phases of cell cycle. (F) Summary of the percentage of cells in SG 2 M from each population. Each point represents a single mouse and bars represent the mean for each population. (G and H) bPTEN +/+ and bPTEN −/− bone marrow was analyzed as in C and D (G), and the percentage of cells in SG 2 M from each population was summarized (H), n = 4. Each point represents a single mouse and bars represent the mean for each population. *, P < 0.05.

Article Snippet: Bone marrow was collected from WT and Ccnd3 −/− mice, erythrocytes lysed, cells suspended in staining buffer (10% [vol/vol] FBS in PBS), and stained ( ) with antibodies against CD3 (17A2), CD8α, CD11b, NK1.1, Ter119, CD43 (BD), CD4 (GK1.5), B220 (RA3-6B2; both from eBioscience), and IgM (115–097-020, Jackson ImmunoResearch Laboratories; or II/41, eBioscience).

Techniques: Cell Culture, Lysis, SDS Page, Flow Cytometry, Expressing