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: International Journal of Molecular Sciences

Article Title: Low-Dose-Rate Radiation-Induced Secretion of TGF-β3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding

doi: 10.3390/ijms23158147

Figure Lengend Snippet: Survival curves for T-47D cells after pretreatment with small extracellular vesicles (sEVs). ( a ) Pretreatment with sEVs from low dose rate (LDR) primed T-47D cells (■) removed the hyper-radiosensitive (HRS) response to subsequent challenge irradiation in T-47D reporter cells. Pretreatment with sEVs from unirradiated controls (►) had no effect on the radiosensitivity of reporter cells. ( b ) Replot from . Previously untreated T-47D cells (▲) exhibit the HRS response to low doses of ionizing radiation (IR). T-47D cells that were primed with a dose of 0.3 Gy delivered at an LDR of 0.3 Gy/h (●) lost HRS. ( c ) Pretreatment with inducible nitric oxide synthase (iNOS) inhibitor 1400 W (◆) or neutralizing antibody to transforming growth factor β3 (TGF-β3) (▼) together with sEVs from LDR primed cells, negated the effect of the sEVs of abolishing the HRS response in reporter cells. LQ model and IR model: linear-quadratic model fit and induced repair model fit, respectively, for untreated T-47D cells. Surviving fractions are given as error-weighted means of three separate experiments, each with five biological replicates. Error bars represent standard error of the mean (SEM). Note that the surviving fractions were calculated relative to the plating efficiency of control cells, which were also exposed to the pretreatments.

Article Snippet: Recombinant TGF-β3, TGF-β3 neutralizing antibody and TGF-βRII neutralizing antibody were purchased from R&D (243-B3, AF-243-NA and AF-241-NA; R&D Systems, Minneapolis, MN, USA), ALK1 inhibitor K02288 from Selleckchem (SMS-group, Rungsted, Denmark) and ALK5 inhibitor SB 431542 and iNOS inhibitor 1400 W from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques: Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Low-Dose-Rate Radiation-Induced Secretion of TGF-β3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding

doi: 10.3390/ijms23158147

Figure Lengend Snippet: Survival curves for T-47D cells after pretreatment with TGF-β3 and inhibitors of relevant receptors. ( a ) As previously demonstrated, pretreatment with TGF-β3 (●) removed the HRS response in T-47D cells. Pretreatments with inhibitors of activin receptor like kinase 5 (ALK5) (◀), TGF-βRII (◆), or ALK2 ( ▼ ) together with TGF-β3 did not influence the ability of TGF-β3 to remove the HRS response. ( b ) Pretreatment with inhibitor of activin receptor like kinase 1 (ALK1) together with TGF-β3 ( ■ ) negated the effect of TGF-β3 alone (●) on the removal of HRS in T-47D cells. LQ model and IR model: linear-quadratic model fit and induced repair model fit, respectively, for untreated T-47D cells. Surviving fractions are given as error-weighted means of three separate experiments, each with five biological replicates. Error bars represent SEM. Note that the surviving fractions were calculated relative to the plating efficiency of control cells, which were also exposed to the pretreatments.

Article Snippet: Recombinant TGF-β3, TGF-β3 neutralizing antibody and TGF-βRII neutralizing antibody were purchased from R&D (243-B3, AF-243-NA and AF-241-NA; R&D Systems, Minneapolis, MN, USA), ALK1 inhibitor K02288 from Selleckchem (SMS-group, Rungsted, Denmark) and ALK5 inhibitor SB 431542 and iNOS inhibitor 1400 W from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Low-Dose-Rate Radiation-Induced Secretion of TGF-β3 Together with an Activator in Small Extracellular Vesicles Modifies Low-Dose Hyper-Radiosensitivity through ALK1 Binding

doi: 10.3390/ijms23158147

Figure Lengend Snippet: Surviving fraction of T-47D cells after 0.2 Gy and pretreatment with inhibitors of different receptors. ( a ) As previously demonstrated, pretreatment with TGF-β3 removed HRS in T-47D cells, shown here by an increase in the surviving fraction after 0.2 Gy γ-irradiation. Pretreatment with inhibitor of ALK5 resulted in a similar effect, both alone and in combination with TGF-β3. ( b ) Pretreatment with inhibitor of ALK1 negated the ability of TGF-β3 to remove HRS in T-47D cells when added together, and did not influence the surviving fraction when added alone. Pretreatment with inhibitor of ALK5 in combination with inhibitor of ALK1 did not influence the latter’s effect on the surviving fraction with or without addition of TGF-β3. ( c ) Removal of HRS by inhibition of ALK5 was negated by combined pretreatment with neutralizing antibody to TGF-β3. ( d ) Pretreatment with iNOS inhibitor 1400 W in combination with either TGF-β3 or inhibitor of ALK5 restored HRS to T-47D cells. Surviving fractions are given as error-weighted means of two (b) or three (a, c, d) separate experiments, each with five biological replicates. Error bars represent SEM. Statistical analysis: One-way ANOVA with post hoc Tukey’s HSD. Note that the surviving fractions were corrected against the plating efficiency of control cells that had not been subjected to pretreatment.

Article Snippet: Recombinant TGF-β3, TGF-β3 neutralizing antibody and TGF-βRII neutralizing antibody were purchased from R&D (243-B3, AF-243-NA and AF-241-NA; R&D Systems, Minneapolis, MN, USA), ALK1 inhibitor K02288 from Selleckchem (SMS-group, Rungsted, Denmark) and ALK5 inhibitor SB 431542 and iNOS inhibitor 1400 W from Sigma-Aldrich (Saint Louis, MO, USA).

Techniques: Irradiation, Inhibition

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: Journal of Clinical Investigation

Article Title: Angiotensin II–dependent TGF-β signaling contributes to Loeys-Dietz syndrome vascular pathogenesis

doi: 10.1172/jci69666

Figure Lengend Snippet: Figure 4 Defective TGF-β receptor signaling in LDS VSMCs in response to exogenous ligand but not under normal culture conditions. (A) Aortic VSMCs derived from wild-type and Tgfbr2G357W/+ mice were starved for 24 hours in 2% serum and then exposed to 10 or 1 ng/ml TGF-β1 for 1 hour. Signaling events were assayed by Western blot (the black line indicates lanes that were run on the same gel but were noncontiguous) (n = 3). (B) Western blot analysis of wild-type and Tgfbr2+/– VSMCs stimulated as in A (n = 3). (C) Western blot analysis of wild-type and 2x Tg-Tgfbr2GW VSMCs stimulated as in A (n = 3). (D) Levels of pSmad2 in unstimulated control and Tgfbr2G357W/+ VSMCs grown in 5% serum to approximately 80% confluence prior to analysis (n = 4). (E) Analysis of Tgfb1, Tgfb2, and Tgfb3 expression in control and Tgfbr2G357W/+ VSMCs cultured as in D (n = 4). The upper and lower margins of the box define the 75th and 25th percentiles, respectively; the internal line defines the median, and the whiskers define the range. *P < 0.05.

Article Snippet: The mouse monoclonal antibody against TGF-β1, TGF-β2, and TGF-β3 (R&D Systems, clone 1D11) was given 3 times per week by intraperitoneal injection (5 mg/kg).

Techniques: Derivative Assay, Western Blot, Control, Expressing, Cell Culture

Journal: Journal of Clinical Investigation

Article Title: Angiotensin II–dependent TGF-β signaling contributes to Loeys-Dietz syndrome vascular pathogenesis

doi: 10.1172/jci69666

Figure Lengend Snippet: Figure 8 Amelioration of LDS vascular pathology by losartan treat- ment correlates with inhibition of pSmad2 and decreased expression of TGF-β ligand. (A) Representative images of pSmad2, pERK1/2, CD45, and smooth muscle myo- sin heavy chain (SMMHC) staining in the aortic roots of 24-week-old Tgfbr2 G357W/+ and control mice treated with placebo, propranolol or losa- rtan. Images were acquired as a tile with a ×25 magni- fication. Scale bar: 100 μm. (B) Expression of Tgfb1, Col1a1, and Serpine1 in aortic root samples from 24-week- old Tgfbr2G357W/+ and control mice treated with placebo (Pla) or losartan (Los) (n = 3). The upper and lower margins of the box define the 75th and 25th percentiles, respectively; the internal line defines the median, and the whiskers define the range. *P < 0.05.

Article Snippet: The mouse monoclonal antibody against TGF-β1, TGF-β2, and TGF-β3 (R&D Systems, clone 1D11) was given 3 times per week by intraperitoneal injection (5 mg/kg).

Techniques: Inhibition, Expressing, Staining, Control

Journal: Reproductive Sciences

Article Title: 1,25 Dihydroxyvitamin D3 Enhances the Antifibroid Effects of Ulipristal Acetate in Human Uterine Fibroids

doi: 10.1177/1933719118812720

Figure Lengend Snippet: List of Antibodies With Used Dilutions.

Article Snippet: Anti-transforming growth factor beta 3 (TGF-β3) , Novus Biologicals, NB600-1531 , Rabbit, polyclonal , WB, 1:500.

Techniques:

Journal: Reproductive Sciences

Article Title: 1,25 Dihydroxyvitamin D3 Enhances the Antifibroid Effects of Ulipristal Acetate in Human Uterine Fibroids

doi: 10.1177/1933719118812720

Figure Lengend Snippet: Human Forward and Reverse Primers Sequences for qRT-PCR.

Article Snippet: Anti-transforming growth factor beta 3 (TGF-β3) , Novus Biologicals, NB600-1531 , Rabbit, polyclonal , WB, 1:500.

Techniques: Sequencing