Fig 1.
Expression of fibroblast growth factor 10 (FGF10) in Graves′ orbitopathy (GO) and in non-GO tissues and cells.
(A) Expression of fibroblast growth factor 10 (FGF10) and FGF receptor 2b (FGFR2b) mRNA in GO and non-GO orbital tissues. (B) Microscopy images showing the expression of FGF10 (green) and live-cell nuclei (4’,6-diamidino-2-phenylindole dihydrochloride; blue) in GO and non-GO orbital tissues. (C) Expression of FGF10 protein in GO and non-GO orbital fibroblasts (OFs). Bar graphs show the relative density of FGF10 normalized to the level of β-actin and are represented as means ± standard deviation. (D) Gene expression of fibronectin, collagen Iα, and α- smooth muscle actin in GO and non-GO OFs. Experiments were performed at least three times using different strains. *p < 0.05, **p < 0.01.
Fig 2.
Effect of transforming growth factor (TGF)-β1 on fibrosis-related protein production in the Graves′ orbitopathy (GO) and non-GO orbital fibroblasts (OFs).
After treatment of the OFs with 5 ng/mL TGF-β1 for 0, 1, 3, 6, 24, 48, and 72 h, the protein expression levels of fibronectin, collagen Iα, α-smooth muscle actin increased in a time-dependent manner. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as mean ± standard deviation. Experiments were performed using at least cells using different strains. *p < 0.05.
Fig 3.
Inhibitory effect of FGF10 on Graves′ orbitopathy (GO) and non-GO orbital fibroblasts.
After treatment with 100 ng/mL of rhFGF10 for 0, 6, 24, and 48 h, fibronectin, collagen IIIa (COL3A) and collagen Iα (COL1A) protein expression was significantly decreased at 24 h or 48 h. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as means ± standard deviation. Experiments were performed using at least three cells from different strains. *p < 0.05.
Fig 4.
Effect of transforming growth factor (TGF)-β1, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α on fibroblast growth factor 10 (FGF10) expression in Graves′ orbitopathy (GO) and non-GO orbital fibroblasts (OFs).
(A) Representative western blot images showing that TGF-β1, IL-1β, and TNF-α increased expression of FGF10 at various time points (0–72 h) in both GO and non-GO OFs. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as means ± standard deviation. (B) Expression levels in GO OFs of secreted FGF10 protein in response to TGF-β1, IL-1β, and TNF-α, using ELISA. Experiments were performed at least three times using different strains. *p < 0.05.
Fig 5.
Effect of upregulation of fibroblast growth factor 10 (FGF10) using recombinant human (rh) FGF10 or blockage of FGF10 by small interfering (si) FGF10 transfection on transforming growth factor (TGF)-β1 induced fibrosis-related protein expression.
The rhFGF10 suppressed the TGF-β1 induced fibronectin, collagen Iα (COL1A), and α-smooth muscle actin (α-SMA) expression in Graves′ orbitopathy (GO) orbital fibroblasts (OFs). Knockdown of FGF10 induced the increase of TGF-β1 induced fibronectin, COL1A, and α-SMA expression in GO OFs. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as mean ± standard deviation. Experiments were performed in at least three cells cultured from different strains. *p < 0.05, **p < 0.01.
Fig 6.
Effect of upregulation of fibroblast growth factor 10 (FGF10) using recombinant human (rh) FGF10 or blockage of FGF10 by small interfering (si) FGF10 transfection on interleukin (IL)-1β or tumor necrosis factor (TNF)-α induced proiflammatory protein expression.
(A) FGF10 did not suppress IL-1β induced IL-6, IL-8, cyclooxygenase (COX)-2, and intercellular adhesion molecule-1 (ICAM-1) in Graves′ orbitopathy (GO) orbital fibroblasts (OFs). However, knockdown of FGF10 induced the increase of IL-1β induced IL-6, IL-8 and COX-2 expression in GO OFs. (B) FGF10 treatment attenuated expression of TNF-a induced IL-6 and COX-2 protein. However, knockdown of FGF10 induced the increase of TNF-α induced IL-6, IL-8, COX-2, and ICAM-1 expression. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as mean ± standard deviation. Experiments were performed in at least three cells cultured from different strains. **p < 0.01.
Fig 7.
Role of fibroblast growth factor 10 (FGF10) in the phosphorylation of signaling molecule, extracellular signal-regulated kinase (ERK).
The increase of phosphorylation of ERK was noted in rhFGF10 treated orbital fibroblasts from Graves′ orbitopathy (GO) and non-GO patients. The phosphorylation of ERK gradually increased and decreased after peaking at 30 minutes of treatment. Bar graphs show the relative density of each protein normalized to the level of β-actin and are represented as mean ± standard deviation. Experiments were performed in at least three cells cultured from different strains. *p < 0.05.