Fig 1: SP induces contraction and upregulates gene expression of collagens and fibrotic markers in quiescent keratocytes. a RT-qPCR showed that COL5A1 gene was upregulated both 4 days and 8 days after treatment with 10-5M SP. Gene expression of LUM was downregulated at day 4 but upregulated 8 days after the treatment. Gene expression of COL1A1 and COL3A1 was upregulated 8 days after treatment; however, it was unaffected 4 days after treatment. b RT-qPCR showed that gene expression of two fibrotic markers: FN and ACTA2 was upregulated both 4 days and 8 days after treatment with 10-5M SP. c Bovine collagen I liquid gel was mixed together with 0.25 × 106 keratocytes and 0.1% FBS. The experimental group gels contained 10-5M SP. Gels consisting only of keratocytes and 0.1% FBS served as controls (ctrl). Gel contraction assay revealed that treatment of keratocytes with 10-5M SP results in increased contraction as soon as 4 h after treatment. Values are means ± SD. n.s. (not significant); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Fig 2: 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 3: The correlation between METTL3 and COL3A1 in breast cancer patients. (A,B) Kaplan–Meier analysis for the OS of METTL3 and COL3A1 in TNBC patients of the TMA sections. (C) The expression of METTL3 and COL3A1 detected by IHC in the representative samples of breast cancer. h8, low expression of METTL3 and high expression of COL3A1. b4, middle expression of METTL3 and COL3A1. h6, high expression of METTL3 and low expression of COL3A1. Original magnification, 200×. (D) Heatmap of the expression level of METTL3 and COL3A1 protein in breast cancer patients. (E,F) Correlation of METTL3 and COL3A1 in human TNBC patients (n = 31) and non-TNBC patients (n = 109), respectively, in TMA sections.
Fig 4: METTL3 down-regulated the expression of COL3A1 by increasing m6A levels. (A) MeRIP-qRT-PCR was used to detect the m6A modification level of COL3A1 in MDA-MB-231 and MDA-MB-468 cells transfected with si-NC or si-METTL3. The relative enrichment fold changes were shown as proportions of control cells enrichment. (B) qRT-PCR was used to detect COL3A1 expression in MDA-MB-231 and MDA-MB-468 cells with METTL3 transient overexpression or the combination of METTL3 overexpression with cycloleucine. 18S was used as an internal control. (C) ELISA was used to detect the secretion level of collagen a1 (III) of the supernatant (left panels), intracellular (middle panels) and total secreted protein (right panels) in MDA-MB-231 and MDA-MB-468 cells with METTL3 transient overexpression or the combination of METTL3 overexpression with cycloleucine. *P < 0.05, **P < 0.01. Error bars represent the mean ± SD of three independent experiments.
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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