Fig. 1: Senescence and fibrosis in Fuchs endothelial corneal dystrophy (FECD) patient specimens.

a Brightfield imaging of normal human cadaveric donor corneas demonstrating normal hexagonal mosaic of CEnCs, whereas corneal specimens from FECD patient display disrupted intercellular borders with presence of rosette formations (white arrow) and guttae (white asterisk), hallmark morphological features of FECD (figure insets = high magnification). b SA-β-Gal staining (green; white arrow) showing presence of senescent cells in FECD specimens compared to the normal tissue. c Increased expression of p21 (green; white arrow), and d p16 (red; white arrow) positive cells indicative of senescence in FECD tissues compared to healthy controls. e α-SMA positive cells (green; white arrow) showing elevated expression in FECD specimens compared to normal controls. Blue: nuclei (Hoechst staining). f Quantitative RT-PCR analysis demonstrating upregulation of endothelial-mesenchymal transition (EMT-red bars), fibrosis (orange bars), extracellular matrix (ECM; green bars), senescence (blue bars) and FECD (purple bar) markers in human FECD specimens normalized to normal donor corneal endothelial cells. Data are presented as mean ± SEM; each dot represents one biological replicate. Statistical analysis: One-way Anova with post-hoc Tukey’s multiple comparison test. Statistical significance: **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bar: a) 100 µm, b–e) 25 µm. Biological replicates: immunostaining (n = 3 per marker) and RT-PCR (n = 5 per gene). Guttae are highlighted in white asterisk. U untreated, AS Acute stress, CS Chronic stress.