Table 1.
Antibodies used in this study.
Table 2.
Primer sequences used in this study.
Fig 1.
Human fibroblast subpopulations in vitro.
Primary fibroblast subpopulations from Tenon’s capsule (hTFs) and fibroblasts from orbital fat (hOFs) were cultured for 1, 3 and 7 days, respectively. div = days in vitro. Bar represents 25 μm.
Fig 2.
Relative proliferation of hTFs and hOFs in response to TGF-β1 and PFD in vitro.
Fibroblasts were treated with TGF-β1 [10 ng/ml], PFD [10−3 mol/l] or the combination of TGF-β1 [10 ng/ml] and PFD [10−3 mol/l] for 48 h under serum-free (A and C) and serum (10% FCS) conditions (B and D) as indicated. NC (proliferation rate of untreated cells) was set to 100%. Data are presented as mean ± SD. The results represent the means of three independent experiments. Level of significances: *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.
Fig 3.
RT-PCR analysis of stimulated (TGF-β1) and suppressed (PFD) primary hTFs.
Cultures of hTFs were treated with TGF-β1 [10 ng/ml], PFD [10−3 mol/l] or the combination of both, TGF-β1 and PFD for 48 h under serum-free culture conditions. NC (expression level of respective gene in untreated cells, dotted line) was set to 1. Data are presented as mean ± SD. The results represent the means of four independent experiments. Level of significances: *p≤0.05; **p≤0.01; ***p≤0.001. Differences between TGF-β1 and TGF-β1+PFD are statistically not significant.
Fig 4.
RT-PCR analysis of stimulated (TGF-β1) and suppressed (PFD) primary hOFs.
Cultures of hOFs were treated with TGF-β1 [10 ng/ml], PFD [10−3 mol/l] or the combination of both, TGF-β1 and PFD for 48 h under serum-free culture conditions. NC (expression level of respective gene in untreated cells, dotted line) was set to 1. Data are presented as mean ± SD. The results represent the means of four independent experiments. Level of significances: *p≤0.05; **p≤0.01; ***p≤0.001. Overall, no statistically significant differences between TGF-β1 and TGF-β1+PFD groups could be observed.
Fig 5.
Immunocytochemical characterization of TGF-β1 induced fibronectin and α-SMA expression in primary hTFs in vitro.
After PFA fixation, cells were incubated with primary antibodies directed against fibronectin and α-SMA. Nuclei were stained by DAPI included in the mounting medium. PFD decreased fibronectin and α-SMA expression. Experiments were carried out four times with similar results. Bar represents 50 μm.
Fig 6.
Immunocytochemical characterization of TGF-β1 induced fibronectin and α-SMA expression in primary hOFs in vitro.
After PFA fixation, cells were incubated with primary antibodies directed against fibronectin and α-SMA. Nuclei were stained by DAPI included in the mounting medium. PFD decreased fibronectin and α-SMA expression. Experiments were carried out four times with similar results. Bar represents 50 μm.
Fig 7.
Western blot analysis of hTFs (A) and hOFs (B) under different culture conditions.
Cell lysates of fibroblast subpopulations were prepared and subjected to Western blot analyses using antibodies against fibronectin, collagen I, collagen VI, α-SMA and β-tubulin as a loading control, as indicated.
Fig 8.
Quantification of Western blot data for hTFs (A) and hOFs (B).
Each column represents the mean ± SD from three independent experiments. * indicates significances obtained by comparison of TGF-β1, PFD and TGF-β1+PFD to untreated cultures. # indicates significances obtained by comparison of TGF-β1-treated cultures and cultures with combined treatment TGF-β1+PFD. Level of significances: *(#)p≤0.05; **(##)p≤0.01; ***(###)p≤0.001. No quantification could be performed for α-SMA in hTFs because of the absence of the protein in control and PFD-treated samples (Fig 7).