Fig 2: Isolation and characterization of human corneal keratocyte (HCK)-, fibroblast (HCF)-, and myofibroblast (HCM)-derived extracellular vesicles (EVs). (A) EV pellets (20 μg protein/lane) were analyzed by western blot to probe for vesicle-associated markers. Representative images are shown for vesicle-associated proteins CD63, CD81, and ITGAV; a negative control (GM130); and proteins associated with corneal wound healing, FN1 and THBS1. (B) EV pellets were analyzed by nanoparticle tracking analysis (NTA), and a size distribution histogram for each EV sample is shown. ((B), inset) Transmission electron microscopy images demonstrating EV morphology (high magnification, 49000x; scale bar = 100 nm). (C) The average particle concentrations (particles x1011/mL); (D) mean particle size (nm); and (E) zeta (ζ) potential of EV pellets were measured using NTA with Zetaview™. Data are shown as mean + SEM; n = 3 independent EV preparations; ns = nonsignificant. One-way ANOVA with Tukey’s post-test.

Fig 3: FN1-EDA protein expression is enhanced in QKO cells. (A, B) Cells were seeded in 12-well plates and on the following day, they were incubated with serum-free media for another 24hours. Whole cell lysates (WCL) and conditioned media (CM) were collected and probed for indicated proteins by immunoblotting. (C–E) Cells were transfected with two rounds of non-targeting siCtrl or siEDA. Approximately 24 h after the second siRNA transfection, transfected cells were serum-starved for another 24 h. WCL and CM were then collected and analyzed as mentioned above. Representative immunoblotting results were shown. Data are mean ± SEM from at least two independent experiments, and each experiment had one or two wells that were treated independently (replicates = 3 or 6). Each dot represents one biological replicate. Statistical significance was determined using a two-tailed t test: *P < 0.05; **P < 0.01, ***P < 0.001.

Fig 4: HGFs cultured with rhPN exhibit increased fibronectin synthesis. (A) HGFs cultured on collagen or collagen + rhPN coated plates for 1 day and 7 days were assessed for gene expressions of FN1. Target gene expression was normalized to 18 S using the ΔΔCt method. Data represents mean fold gene expressions ± s.d. relative to control day 1 (on collagen alone) of 3 independent experiments in triplicates. Data was analyzed via Student’s t-test (unpaired) within each time-point (**p < 0.01; ns, not significant). (B) Fibronectin (FN) protein levels were assessed using western blots in cell lysates when HGFs are cultured on collagen (Col) or collagen + rhPN (rhPN) for 1 day and 7 days. GAPDH was used as a loading control. (C) Fibronectin protein detection in the supernatants from HGFs cultured on Col or rhPN for 1 day using western blot. (D,E) HGFs cultured on Col or rhPN coated plates for 14 days were assessed for fibronectin protein level. TGF-β1 treatment of HGFs on collagen-coated plates served as a positive control. (D) Fibronectin protein levels were assessed using western blots in cell lysates (D) when HGFs are cultured for 14 days and in supernatant (E) obtained from the last day of the culture. GAPDH was used as a loading control for cell lysates. Bi,Ci,Di,Ei. Densitometry analyses of the fibronectin bands were performed. For proteins from cell lysates, fibronectin bands were normalized to corresponding GAPDH bands. Data represents mean fold band intensities ± s.d. relative to control day 1 (collagen alone) of 3 independent experiments in triplicates. Data was analyzed via Student’s t-test (unpaired) within each time-point (*p < 0.05; ns, not significant) (Bi-Ci) and one-way ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001) (Di-Ei).

Fig 5: Effect of MDL-800 on TGF-β1-induced β-catenin acetylation and β-catenin target gene promoter enrichment at the site of β-catenin acetylation in HK2 cells. (A) Representative Western blot for acetyl-β-catenin and active-β-catenin from HK-2 cells treated with Veh or TGF-β1 (10 ng/mL), with or without MDL-800 at indicated doses (10, 25, or 50 μM). Treatment with TGF-β1 (10 ng/mL) over 24 h increased the expression of acetyl-β-catenin and active-β-catenin. The expression of acetyl-β-catenin and active-β-catenin decreased in a dose-dependent manner after the administration of MDL-800 treatment. The bar graph shows the densitometric quantification presented as the relative ratio of each protein to GAPDH. Data are presented as mean ± SD. (B) Representative Western blot analysis of β-catenin and Sirt6 from HK-2 cells after Veh or TGF-β1 (10 ng/mL), with or without MDL-800 (50 μM). HK-2 cell lysates were immunoprecipitated against the Sirt6 antibody, and immunoprecipitants were immunoblotted with β-catenin. (C) Chromatin immunoprecipitation (ChIP) assay of β-catenin target genes, such as Fn1, MMP7, and Snail promoter regions in HK2 cells, after 24 h treatment of vehicle (Veh), TGF-β1 (10 ng/mL) with or without MDL-800 (50 μM). Immunoprecipitation was performed using acetyl- β-catenin antibody. Normal mouse IgG was used as a negative control and the PCR product of input chromatin (input 5%) before immunoprecipitation was used as a positive control. Quantitative real-time PCR was performed using specific primers for Fn1, MMP7, and Snail promoter regions. All results were normalized to input levels. Data are expressed as mean ± SD from four independent experiments. ***, p < 0.001 versus Veh or MDL; ###, p < 0.001 versus TGF-β1 (10 ng/mL); ##, p < 0.01 versus TGF-β1 (10 ng/mL). Veh, vehicle; MDL, MDL-800; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin; CTGF, connective tissue growth factor; Fn1, fibronectin; MMP-7, matrix metalloproteinase-7.