Journal: Scientific reports

Article Title: Peripheral serotonin regulates postoperative intra-abdominal adhesion formation in mice.

doi: 10.1038/s41598-017-10582-w

Figure Lengend Snippet: Figure 7. Serotonin facilitated postoperative intra-abdominal adhesion formation by up-regulating TGF- β1 expression. Postoperative intra-abdominal adhesion formation was induced by caecum rubbing, and the mice were sacrificed on the 7th day after the operation. The adhesive tissues were harvested. (A,B) Immunohistochemistry was performed to evaluate the levels of TGF-β1, COX-2, TIMP-1 and MMP-2 in the adhesive tissues (magnification ×200); (C,D) Western blot analysis was conducted to evaluate the levels of 5-HT2B receptor in the adhesive tissues; n = 6, mean ± SEM, #P < 0.05 vs. the WT sham group, *P < 0.05 vs. the WT group.

Article Snippet: The primary antibodies anti- CD34 antibody (bs-9752R, BIOSS, China, 1:300 dilution), anti- VEGF antibody (bs-0279R, BIOSS, China, 1:300 dilution), anti-TGF-β1 antibody (sc-146, Santa Cruz Biotechnology, USA, 1:400 dilution), anti-COX-2 antibody (bs-10411R, BIOSS, China, 1:300 dilution), anti-TIMP-1 antibody (bs-4600R, BIOSS, China, 1:300 dilution) and anti-MMP-2 antibody (bs-4599R, BIOSS, China, 1:300 dilution) were incubated overnight at 4 °C, and a secondary antibody was incubated for 1 h at room temperature.

Techniques: Expressing, Adhesive, Immunohistochemistry, Western Blot

Journal: Scientific reports

Article Title: Peripheral serotonin regulates postoperative intra-abdominal adhesion formation in mice.

doi: 10.1038/s41598-017-10582-w

Figure Lengend Snippet: Figure 9. Peripheral serotonin regulated postoperative intra-abdominal adhesion formation in mice. After the surgery, serum serotonin was substantially released. Serotonin facilitated the following functions: recruitment of inflammatory cells and fibroblasts to the injury site, damage related to oxidative stress to the endotheliocyte, and the disorder of the fibrinolytic system, all of which resulted in collagen deposition, angiogenesis via the up- regulation of VEGF expression, and the secretion of extracellular matrix (ECM) via the up-regulation of TGF- β1 expression. The above mechanisms contributed to postoperative intra-abdominal adhesion formation.

Article Snippet: The primary antibodies anti- CD34 antibody (bs-9752R, BIOSS, China, 1:300 dilution), anti- VEGF antibody (bs-0279R, BIOSS, China, 1:300 dilution), anti-TGF-β1 antibody (sc-146, Santa Cruz Biotechnology, USA, 1:400 dilution), anti-COX-2 antibody (bs-10411R, BIOSS, China, 1:300 dilution), anti-TIMP-1 antibody (bs-4600R, BIOSS, China, 1:300 dilution) and anti-MMP-2 antibody (bs-4599R, BIOSS, China, 1:300 dilution) were incubated overnight at 4 °C, and a secondary antibody was incubated for 1 h at room temperature.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice were sacrificed on 10 dpf. ( A ) Heatmap of the expression of SASP factors in callus as determined by qPCR. n = 3. ( B ) Expression of SASP factors in callus and nonfractured bone was assessed by qPCR. n = 4. Two-way ANOVA followed by Tukey’s post hoc test. ( C ) p16 + SCs isolated from young and aged callus. Expression of SASP factors examined by qPCR. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( D ) p16 + , TGF-β1 + , and p16 + TGF-β1 + cells were identified by flow cytometry. ( E ) Fold changes in p16 + , TGF-β1 + , and p16 + TGF-β1 + cell percentages in callus and nonfractured tibiae of aged versus young mice. n = 4–6. * P < 0.05, for callus versus bone, by unpaired, 2-tailed Student’s t test. ( F ) Percentage of p16 + TGF-β1 + cells in callus and nonfractured tibiae. * P < 0.05; ^ P < 0.05; # P < 0.05, for aged versus young, by 2-way ANOVA followed by Tukey’s post hoc test. ( G ) Expression of TGF-β1 in callus tissues following D+Q treatment by as determined by Western blotting. ( H ) CaMPCs were treated for 2 days with CM from young or aged callus with or without TGF-β–neutralizing Ab or IgG. Cell growth and proliferation were assessed by methylene blue staining or a BrdU incorporation assay. n = 4 wells. * P < 0.05, for IgG versus anti–TGF-β Ab; # P < 0.05, for aged anti–TGF-β Ab versus young IgG, by 2-way ANOVA followed by Tukey’s post hoc test. ( I ) Concentration of active TGF-β1 in CM from young and aged callus cultures measured by ELISA. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( J ) Callus pieces were harvested from young and aged mice and infected with Tgfb1 or scrambled (Ctl) shRNA lentivirus. CM was collected. The expression of Tgfb1 was measured by qPCR. CaMPCs were treated with the CM. Cell growth was assessed using a CCK8 kit. n = 3 wells. * P < 0.05, for control versus Tgfb1 shRNA, by 2-way ANOVA followed by Tukey’s post hoc test. The experiment was repeated once.

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Isolation, Flow Cytometry, Western Blot, Staining, BrdU Incorporation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection, shRNA, Control

Journal: The Journal of Clinical Investigation

Article Title: Age-associated callus senescent cells produce TGF- β 1 that inhibits fracture healing in aged mice

doi: 10.1172/JCI148073

Figure Lengend Snippet: Young and aged mice underwent tibial fracture surgery. ( A ) The expression of Tgfb1 in fracture callus at indicated time points was measured by qPCR. n = 3. Relative mRNA expression is the fold-change versus young mice as 1. ( B ) The concentration of active TGF-β1 protein in fracture callus at indicated time points was measured by ELISA. n = 4. * P < 0.05, for aged versus young; # P < 0.05, for young versus young 0 dpf; ^ P < 0.05, for aged versus aged 0 dpf, by 2-way ANOVA followed by Tukey’s post hoc test ( A and B ). ( C ) Outline of the experimental design. Aged mice were given 2 μg in 10 μL TGF-β Ab, 1D11, or isotype IgG vehicle by intra-callus injection on 1, 3, 5, and 7 dpf and sacrificed on 10 dpf ( D – F and H – J ) or 28 dpf ( G ). n = 4–5. ( D ) Callus volume was measured by micro-CT. * P < 0.05, by unpaired, 2-tailed Student’s t test. ( E ) Representative images of ABH-stained sections showing more woven bone and callus areas in the anti–TGF-β Ab–treated mice. Scale bar: 1 mm. ( F ) Woven bone and cartilage areas were analyzed using Visiopharm software. ( G ) Bone stiffness, strength and toughness were assessed by biomechanical testing at 28 dpf. ( H ) The percentage and number of MPCs identified as CD45 – CD31 – CD105 + cells in fracture callus were determined by flow cytometry. ( I ) Representative paraffin sections of callus immunostained with anti-Ki67 Ab to detect proliferating cells (arrowheads). External callus is indicated by the dashed lines. Scale bars: 500 μm. Original magnification, ×4 (enlarged insets). ( J ) The percentage of Ki67 + cells was quantified by ImageJ. * P < 0.05, by unpaired, 2-tailed Student’s t test ( F , G , H , and J ).

Article Snippet: The shRNA lentiviral particles targeting Tgfb1 (sc-37192-V) and a scrambled sequence (sc-108080) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Injection, Micro-CT, Staining, Software, Flow Cytometry