1. Thy-1/β3 Integrin Interaction-Induced Apoptosis of Dermal Fibroblasts Is Mediated by Up-Regulation of FasL Expression 
Manuela Schmidt, Danny Gutknecht, Konstantinos Anastassiadis, Beate Eckes, Ulf Anderegg, Anja Saalbach 
Journal of Investigative Dermatology 
Volume 136, Issue 2, Pages (February 2016)
DOI: /j.jid
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2. Figure 1
Thy-1–mediated apoptosis in dermal fibroblasts is mediated by up-regulation of FasL expression. Primary skin fibroblasts were enzymatically isolated from complete Thy-1–deficient (Thy-1–/–) and wild-type (wt) control mice and analyzed for expression of Fas ligand (FasL) and Fas receptor. (a) FasL mRNA and (b) protein expression level of wt and Thy-1–/– fibroblasts at different time points analyzed by quantitative polymerase chain reaction and flow cytometry (fibroblasts of n ≥4 animals per genotype). (c) Fas receptor mRNA and (d) protein expression level of wt and Thy-1–/– fibroblasts (fibroblasts of n ≥4 animals per genotype). (e, f) Primary wt and Thy-1–/– fibroblasts were seeded for 72 hours in the presence or absence of function-blocking anti-CD61 (β3 integrin) or anti-CD29 (β1 integrin) antibody and appropriate isotype control. (e) FasL mRNA expression was detected by quantitative polymerase chain reaction (fibroblasts of n = 3 animals per genotype). (f) Representative images of FasL protein expression detected by immunofluorescence staining (red). Cell nuclei were counterstained with DAPI (blue). Bar = 50 μm. (g–i) Cell proliferation (after 24 hours), caspase 3/7 activity (after 72 hours), and cell growth (after 72 hours) of wt and Thy-1–/– fibroblasts in the presence or absence of function-blocking anti-FasL antibody and isotype control (fibroblasts of n = 4 animals per genotype). Data are given as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; n.s., not significant; RLU, relative light units.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions

3. Figure 2
Thy-1–mediated apoptosis in granulation tissue and wound fibroblasts by up-regulation of FasL expression. Full-thickness wounds were induced under anesthesia on the backs of conditional fibroblast-specific knockout (Col1-Thy-1) and Thy-1fl/fl, Cre–/– control mice. (a) Double staining of CD61 (β3 integrin, red) and Thy-1 (green) as well as (b) Fas ligand (FasL, red) and Thy-1 (green) in granulation tissue of 9-day-old wounds of Thy-1fl/fl, Cre–/– control mice. Cell nuclei were counterstained with DAPI (blue). White arrows mark double-positive cells (yellow) in merged images. Bar = 50 μm. (c) Representative images of FasL and (d) Fas expression in granulation tissue of 9-day-old wounds from conditional fibroblast-specific Col1-Thy-1 and Thy-1fl/fl, Cre–/– control mice, analyzed by immunofluorescence staining. Cell nuclei were counterstained with DAPI (blue). Bar = 50μm. (e–i) Primary wound fibroblasts of conditional fibroblast-specific Col1-Thy-1 and Thy-1fl/fl, Cre–/– control mice were isolated by enzymatic digestion. mRNA expression level of (e) FasL and (f) Fas were analyzed by quantitative polymerase chain reaction after 72 hours (wound fibroblasts of n = 4 animals per genotype). (g) Cell growth (after 72 hours), (h) caspase 3/7 activity (after 72 hours) and (i) proliferation rate (after 24 hours) of isolated wound fibroblasts (wound fibroblasts of n = 8 animals per genotype). Data are given as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P≤ (j) Schematic overview for Thy-1–mediated regulation of fibroblast growth: (1) Thy-1 interacts with β3 integrins on fibroblast cell surface and (2) induces up-regulation of FasL. Binding to Fas receptor (3) results in extrinsic activation of caspase cascade indicated by enhanced activity of both effector caspases 3 and 7, which leads to increased apoptosis. In parallel, proliferation of β3 integrin–expressing cells is suppressed. The combination of less proliferation and increased apoptosis results in overall decreased cell growth of Thy-1–expressing fibroblasts. (4) An additional regulation pathway of fibroblast proliferation is suggested by the different time points of Thy-1–mediated effects on proliferation and apoptosis. (5) Furthermore, Thy-1 supports differentiation of fibroblasts to myofibroblasts via a still unknown ligand. BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; gt, granulation tissue; he, hyperproliferative epidermis; n.s., not significant; RLU, relative light units.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2015 The Authors Terms and Conditions