Fig. 5: Epigenetic regulation of α-Klotho by stiff matrix disrupts chondrocytes health.

A Stiff substrates downregulated Klotho expression and increased Klotho promoter methylation as well as global DNA methylation in young chondrocytes (n = 3/group). B Stiff substrates increased DNMT1 expression in young chondrocytes as quantified by immunofluorescence (n = 5/group; 30–50 cells per individual sample). Scale bar: 20 μm. C Stiff substrates increased binding of DNMT1 at Klotho promoter in young chondrocytes quantified by chromatin immunoprecipitation (ChIP) analyses (n = 4/group). D Stiff substrates increased binding of RNA Polymerase II (Pol II), active chromatin mark H3K4M2, and c-MYC, but not the repressive chromatin mark, H3K9M2, at the Dnmt1 promoter in young chondrocytes, as quantified by ChIP analyses (n = 4/group). E, F siRNA Dnmt1 treatment inhibited the deleterious effect of a stiff microenvironment on α-Klotho, type II collagen (Col2), and aggrecan (Acan) expression, as quantified by immunofluorescence (n = 4/group; 40–50 cells per individual sample). Scale bar: 50 μm. G, H 5-Aza-2’-deoxycytidine (5 Aza) treatment inhibits the deleterious effect of a stiff microenvironment on α-Klotho, Col2, and Acan expression. The effects of 5 Aza treatment were blocked when combined with siRNA Klotho (siRNA KL) treatment. Data were quantified by immunofluorescence (n = 4/group; 30–45 cells per individual sample). Scale bar: 50 μm. Statistical analyses were performed using a linear mixed effect model (A–D), two-tailed paired t test (E–G) or analysis of variance with post-hoc Tukey–Kramer test (H). **p < 0.01, ***p < 0.001. Data are presented as means ±95% confidence intervals. Source data are provided as a Source Data file.