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.