Fig 1: In vitro human corneal fibrosis model. (A) qRT-PCR analysis showing RNA expression levels of COL1A1, COL3A1, COL5A1, LUM, FN and ACTA2 after induction of fibrosis in human corneal fibroblasts. Each time point was compared to time 0 (set to fold 1). (B) Secretion of pro-collagen I, collagen III, collagen V, lumican and fibronectin after induction of fibrosis in human corneal fibroblasts was assessed by ELISA. (C) Western blot analysis of a-SMA (42 kDa) expression after induction of fibrosis in human corneal fibroblasts. ß-Actin (45 kDa) served as loading control. Densitometry analysis was performed with ImageJ software. (D) Collagen gel contraction assay was used to determine the contractile abilities of the cells after induction of fibrosis in human corneal fibroblasts. Values are means ± SD. n.s. (not significant); *P < .05; **P < .01; ***P < .001; ****P < .0001
Fig 2: ACh decreases gene expression and production of extracellular matrix components and fibrotic markers in quiescent keratocytes. (A) qRT-PCR analysis in human primary keratocytes showing RNA expression levels of COL1A1, COL3A1, COL5A1, LUM, FN and ACTA2 after ACh stimulation. Each time point was compared to time 0 (set to fold 1). (B) Secretion of pro-collagen I, collagen III, lumican and fibronectin after ACh treatment was assessed by ELISA. (C) Western blot analysis showing the effect of ACh on a-SMA (42 kDa) expression in human primary keratocytes. ß-Actin (45 kDa) served as loading control. (D) Scratch assay showing % of closed wound after ACh stimulation at specific time points. Densitometry analysis and scratch assay evaluation were performed with ImageJ software. Values are means ± SD. n.s. (not significant); *P < .05; **P < .01; ***P < .001; ****P < .0001
Fig 3: ACh decreases expression and production of extracellular matrix components during the process of fibrosis. (A) Effect of ACh on RNA expression levels of COL1A1, COL3A1, COL5A1 and LUM 2 and 4 days after induction of fibrosis in human corneal fibroblasts, as assessed by qRT-PCR. Each time point was compared to time 0 (set to fold 1). (B) Effect of ACh and atropine on secretion of pro-collagen I, collagen III, collagen V and lumican after induction of fibrosis in human corneal fibroblasts was determined by ELISA. Values are means ± SD. n.s. (not significant); *P < .05; **P < .01; ***P < .001; ****P < .0001
Fig 4: ACh decreases gene expression and production of fibrotic markers in persistent fibrosis. (A) Effect of ACh on RNA expression levels of ACTA2 and FN in persistent fibrosis setting 2 days, 4 days, 6 days, and 8 days after ACh treatment, assessed by qRT-PCR. (B) Effect of ACh on RNA expression levels of COLA1A, COL3A1 and COL5A1 in persistent fibrosis setting 2, 4, 6 and 8 days after ACh treatment, assessed by qRT-PCR. (C) Cumulative secretion of fibronectin and pro-collagen I after ACh treatment of persistent fibrosis for a period of 8 days, determined by ELISA. Values are means ± SD. n.s. (not significant); *P < .05; **P < .01; ***P < .001; ****P < .0001
Fig 5: Changes in various ECM components in corneal myofibroblasts are dependent on mitochondrial ROS and NF-?B signaling. (A) RT qPCR on the mRNA expression of COL1A1, COL3A1, COL5A1 and lumican in corneal myofibroblasts (n = 3). (B) Secretion of pro-collagen I, collagen III, collagen V, and hydroxyproline content in supernatant collected 2 d after fibrosis induction (n = 3). (C) RT qPCR of the mRNA expression of COL1A1, COL3A1, COL5A1 and lumican in CD+ corneal myofibroblasts. Cells were treated with 10 µM mitoTEMPO for 2 d (n = 3). (D) Secretion of pro-collagen I, collagen III, collagen V, and hydroxyproline content in supernatant collected 2 d after fibrosis induction in CD+ myofibroblasts simultaneously treated with 10 µM mitoTEMPO (n = 3). (E) RT qPCR of the mRNA expression of COL1A1, COL3A1, COL5A1 and lumican in CD+ corneal myofibroblasts treated with 20 nM TPCA for 48 h (n = 3). (F) Secretion of pro-collagen I, collagen III, collagen V, and hydroxyproline content in supernatant collected 2 days after fibrosis induction in CD+ myofibroblasts simultaneously treated with 20 nM TPCA (n = 3). Values are means ± SD. n.s. (not significant); *P < 0.05, **P < 0.01.
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