Journal: Scientific Reports

Article Title: Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling

doi: 10.1038/s41598-017-12919-x

Figure Lengend Snippet: ASC-derived exosomes increased the ratio of TGF-β3 to TGF-β1 in vivo . ( A–D ) Representative images of immunohistochemical staining of TGF-β1, TGF-β3 and CD63 at days 3, 7, 14 and 21 following treatment with 200 µg ASC-Exos or the same volume of PBS orexosome-free conditioned medium (CM-Exo). Scale bar = 100 µm. ( E ) Analysis of the relative density of TGF-β1, TGF-β3 and CD63. Results are presented as mean ± SD; n = 5; *p < 0.05, ** p < 0.01, ***p < 0.001, compared with the control groups.

Article Snippet: Samples were incubated with primary antibodies against CollagenI(1:100, Abcam), Collagen III(1:100, Abcam), TGF-β1 (1:200, Proteintech), TGF-β3 (1:200, Proteintech) and CD63 (1:200, Proteintech) overnight at 4 °C.

Techniques: Derivative Assay, In Vivo, Immunohistochemical staining, Staining, Control

Journal: Scientific Reports

Article Title: Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling

doi: 10.1038/s41598-017-12919-x

Figure Lengend Snippet: ASC-derived exosomes affected the expression of genes associated with scarless wound healing in dermal fibroblasts. ( A ) mRNA expression of α-SMA , COL1A1 , COL3A1 , MMP1 , MMP3 , TIMP1 , TGF-β1 and TGF-β3 in human fibroblasts treated with 0, 25, 50 or 100 µg/mL ASC-Exos for 24 hours. ( B ) Western blot analysis of the expression of α-SMA, Collagen I, Collagen III, MMP1, MMP3, TIMP1, TGF-β1and TGF-β3 in fibroblasts treated with ASC-Exos (100 µg/mL) for 24 hours. ( C ) Densitometric analysis of the western blot bands. Results are presented as mean ± SD; n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control groups.

Article Snippet: Samples were incubated with primary antibodies against CollagenI(1:100, Abcam), Collagen III(1:100, Abcam), TGF-β1 (1:200, Proteintech), TGF-β3 (1:200, Proteintech) and CD63 (1:200, Proteintech) overnight at 4 °C.

Techniques: Derivative Assay, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling

doi: 10.1038/s41598-017-12919-x

Figure Lengend Snippet: ASC-derived exosomes contributed to regulating ECM reconstruction and reducing scar formation. Exosomes increased the ratio of collagen III to collagen I, reduced myofibroblast differentiation and increased the ratio of TGF-β3 to TGF-β1 in vivo . In addition, exosomes activated the ERK/MAPK pathway in skin dermal fibroblasts and increased the level of MMP3 via activation of the ERK pathway, thereby increasing the ratio of MMP3 to TIMP1 and regulating ECM remodelling.

Article Snippet: Samples were incubated with primary antibodies against CollagenI(1:100, Abcam), Collagen III(1:100, Abcam), TGF-β1 (1:200, Proteintech), TGF-β3 (1:200, Proteintech) and CD63 (1:200, Proteintech) overnight at 4 °C.

Techniques: Derivative Assay, In Vivo, Activation Assay

Journal: Scientific Reports

Article Title: Exosomes secreted by human adipose mesenchymal stem cells promote scarless cutaneous repair by regulating extracellular matrix remodelling

doi: 10.1038/s41598-017-12919-x

Figure Lengend Snippet: Primers of quantitative reverse transcription–polymerase chain reaction (qRT-PCR).

Article Snippet: Samples were incubated with primary antibodies against CollagenI(1:100, Abcam), Collagen III(1:100, Abcam), TGF-β1 (1:200, Proteintech), TGF-β3 (1:200, Proteintech) and CD63 (1:200, Proteintech) overnight at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation.

doi: 10.1210/endo.141.4.7419

Figure Lengend Snippet: FIG. 1. Characterization of TGFb-IR in the anterior pituitary. This is a representative photograph showing that TGFb3 (blackish blue) cells also contain PRL (brown) in anterior pituitary tissue obtained from a cyclic (C) female rat on the day of estrus. Tissue was pro- cessed for immunostaining using TGFb3, PRL antibodies, and dou- ble immunohistochemical procedures. Arrowheads indicate some cells positive for both PRL and TGFb3. A corresponding photo- graph showing colocalization of TGFb1 and PRL has been pub- lished by us previously (6).

Article Snippet: The membranes were then placed in 5% milk block for 5 h, followed by incubation in primary antibody (monoclonal TGFb3 antibody, R&D Systems, Minneapolis, MN; 1 mg/ml) in blocking buffer at 4 C overnight.

Techniques: Immunostaining, Immunohistochemical staining

Journal: Endocrinology

Article Title: Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation.

doi: 10.1210/endo.141.4.7419

Figure Lengend Snippet: FIG. 2. Effect of estradiol on TGFb3-IR in anterior pituitary tissue. A, Representative photographs show TGFb3 immunoreactivity (brown) in a 2-mm section from the anterior pituitary from an estradiol-treated rat (4-week treatment; left) and from an ovariectomized rat (4 weeks posttreatment; middle). The sections were counterstained with Gill’s hematoxylin, which accounts for the blue nuclear staining in each cell. A pituitary tissue section of an estradiol-treated rat showed reduced staining when this section was treated with antigen-preincubated TGFb3 antibody (right). Scale bar, 20 mm. B, Mean 6 SE percentage of cells reacted with TGFb3 antibody in anterior pituitary of ovariectomized (OVEX; 4-week treatment) rats and estradiol-treated OVEX rats (E2; 4-week treatment). a, P , 0.05. n 5 4 rats/group.

Article Snippet: The membranes were then placed in 5% milk block for 5 h, followed by incubation in primary antibody (monoclonal TGFb3 antibody, R&D Systems, Minneapolis, MN; 1 mg/ml) in blocking buffer at 4 C overnight.

Techniques: Staining

Journal: Endocrinology

Article Title: Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation.

doi: 10.1210/endo.141.4.7419

Figure Lengend Snippet: FIG. 3. Characterization of TGFb3 and TGFb1 mRNA levels in response to es- tradiol and ovariectomy. TGFb3 and TGFb1 mRNA contents were measured in the anterior pituitary by Northern blot analysis as described previously (7). A, Representative Northern blot au- toradiographs showing the changes in TGFb1 and TGFb3 mRNA contents in the anterior pituitaries from cyclic fe- male rats on the day of estrus (C), ovari- ectomized rats treated for 4 weeks with estradiol (E), or ovariectomized rats not treated with estradiol (O). Eighteen mi- crograms of total RNA from each pitu- itary sample were electrophoresed through a 1.3% agarose gel and trans- ferred to nylon filters. Blots were hy- bridized with 32P-labeled probes for rat TGFb1, mouse TGFb3, and human 18S RNA. Blots were autoradiographed for 24–48 h at 270 C. B and C, Summary of mRNA quantification data carried out by densitometric analysis of the au- toradiogram by laser scanner. The val- ues were normalized to the 18S RNA. n 5 5–6 rats/group.

Article Snippet: The membranes were then placed in 5% milk block for 5 h, followed by incubation in primary antibody (monoclonal TGFb3 antibody, R&D Systems, Minneapolis, MN; 1 mg/ml) in blocking buffer at 4 C overnight.

Techniques: Northern Blot, Agarose Gel Electrophoresis, Labeling

Journal: Endocrinology

Article Title: Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation.

doi: 10.1210/endo.141.4.7419

Figure Lengend Snippet: FIG. 4. Western blot analysis of TGFb3 protein in anterior pituitary tissue of cyclic (on the day of estrus), ovariecto- mized (4 weeks postovariectomy), and estradiol-treated (4-week treatment) rats. Pituitaries were homogenized, and samples were acid activated and neutralized before loading. One hun- dred micrograms of protein were loaded from each sample. A, A representative photograph of a Western blot incubated with antibody specific for TGFb3 (0.2 mg/ml). Lane A, Estradiol; lane B, cyclic; lane C, ovariectomized; lane D, 10 ng recombinant human TGFb3. B, The same membrane as that blotted in A was stained with Ponceau S solution to check transfer efficiency and equal protein loading. C, The relative pro- tein amount in each band was deter- mined by densitometric analysis of the enhanced chemiluminescence-exposed film by laser scanner. For each blot the density of the band corresponding to TGFb3 in the treatment groups was compared with that in the control (cy- clic) tissue. a, P , 0.05. n 5 6 rats/group.

Article Snippet: The membranes were then placed in 5% milk block for 5 h, followed by incubation in primary antibody (monoclonal TGFb3 antibody, R&D Systems, Minneapolis, MN; 1 mg/ml) in blocking buffer at 4 C overnight.

Techniques: Western Blot, Incubation, Recombinant, Membrane, Staining, Control

Journal: Endocrinology

Article Title: Opposing actions of two transforming growth factor-beta isoforms on pituitary lactotropic cell proliferation.

doi: 10.1210/endo.141.4.7419

Figure Lengend Snippet: FIG. 5. TGFb1 and TGFb3 regulation of lactotropic cell proliferation. Primary cultures of anterior pituitary cells were prepared as described in Materials and Methods. A–E, Photographs show PRL-stained (red), BrdUrd-stained (brown), and hematoxylin-stained (blue) cells in representative cultures treated with vehicle (A; 4 mM HCl and 1% BSA; control), estradiol (B; 10 nM), estradiol and TGFb1 (C; 10 nM estradiol and 10 ng/ml TGFb1), estradiol and TGFb3 (D; 10 nM estradiol and 10 ng/ml TGFb3), and estradiol and anti-TGFb3 (E; 10 nM estradiol and 10 mg/ml TGFb3 antibody). Arrowheads indicate some double stained cells. Cells were plated for 4 days in culture, then treated for 4 days with the various peptides and/or antibodies. Four hours before fixation of the cells, cultures received 0.1 mM BrdUrd, and using double immuno- cytochemistry, we determined the percentage of lactotropes proliferating by colocalizing BrdUrd and PRL immunoreactivities in a single cell. Bar, 20 mm. F, Graph depicting the dose-response effects of TGFb1 and TGFb3 in estradiol’s presence and absence on lactotropic proliferation. n 5 5–12/group. ANOVA indicated significant dose-response effects for TGFb3 (F 5 5.1; P , 0.004), TGFb1 with estradiol (F 5 3.9; P , 0.01), TGFb3 with estradiol (F 5 7.2; P , 0.0005), and TGFb1 antibody with TGFb3 (F 5 3.7; P , 0.02). There was a significant difference between groups treated with TGFb3 alone and those treated with TGFb3 and estradiol (P , 0.001 for each dose of TGFb3). Immunoneutralization of TGFb1 significantly increased the ability of TGFb3 to stimulate lactotropic cell proliferation compared with that of TGFb3 alone (P , 0.01 for all TGFb3-treated groups). G, Histogram summarizing the effects of anti-TGFb1 (10 mg/ml) and anti-TGFb3 (10 mg/ml) on estrogen’s ability to induce proliferation in lactotropes. 17b-Estradiol (10 nM) was used. The control and estradiol groups received 10 mg/ml rabbit g-globulin (Calbiochem, La Jolla, CA). n 5 5–6/group.

Article Snippet: The membranes were then placed in 5% milk block for 5 h, followed by incubation in primary antibody (monoclonal TGFb3 antibody, R&D Systems, Minneapolis, MN; 1 mg/ml) in blocking buffer at 4 C overnight.

Techniques: Staining, Control, Immunocytochemistry

Journal: PLoS Genetics

Article Title: Overexpression of Latent TGFβ Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFβ

doi: 10.1371/journal.pgen.1006019

Figure Lengend Snippet: (A) Quadriceps muscles from 8-week old animals were sectioned and stained with hematoxylin and eosin, showing improved histopathology of TG+ mdx mice compared to mdx controls. The region selected for imaging was selected from the image in (boxed). (B) The fibrosis content of TG+ mdx animals was assessed at 20 weeks using an assay for hydroxyproline, a modified amino acid found in collagen. Shown is hydroxyproline content from quadriceps, gluteus/hamstrings and triceps muscles, each showing reduction in hydroxyproline content in TG+ mdx (n = 7) compared to mdx (n = 8). (C) HEK293T cells were transfected to express heterologously mouse LTBP4 and either TGFβ1, TGFβ2, or TGFβ3. Co-immunoprecipitation was performed on cell lysates by immunoprecipitating with anti-LTBP4 antibody followed by immunoblotting with anti-myc antibody detecting TGFβ1, 2, and 3. A co-IP control for each experiment was performed without adding IP antibody. LTBP4 associated with all three TGFβ isoforms in vitro. Scale bar = 100μm.

Article Snippet: Mouse cDNA clones of Mstn (NM_010834), Gdf11 (NM_010272), Tgfβ1 (NM_011577), Tgfβ2 (NM_009367), and Tgfβ3 (NM_009368) were purchased from Origene (catalog numbers MR227629, MR223819, MR227339, MR225633, and MR206441, respectively).

Techniques: Muscles, Staining, Histopathology, Imaging, Modification, Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Control, In Vitro

Journal: Cell Transplantation

Article Title: Ideal Duration of Pretreatment Using a Gelatin Hydrogel Nonwoven Fabric Prior to Subcutaneous Islet Transplantation

doi: 10.1177/09636897231186063

Figure Lengend Snippet: List of Analyzed Target Genes.

Article Snippet: Mm00436960_m1 , Tgfb3 , Transforming growth factor beta 3.

Techniques: Derivative Assay