Fig. 3

Chondrocytes iron overload could disrupt mitochondria and activate the mtDNA/cGAS/STING pathway. (A) Chondrocytes were treated with increasing concentrations of FAC and expressions of cGAS and STING were examined by western blotting. GAPDH was included as a loading control and semi-quantitative analysis of band density was conducted. (B) Representative images of IF staining for STING expression in chondrocytes treated with FAC for 24 h. Scale bars = 50 μm. (C) Chondrocytes were treated with 5ng/ml IL-1β for 12 h with or without TfR1 siRNA, then expressions of cGAS and STING were examined by western blotting. GAPDH was included as a loading control and semi-quantitative analysis of band density was conducted. (D) JC-1dye immunofluorescence staining was conducted to detect the mitochondrial membrane potential. Scale bars = 200 μm. (E) Representative fluorescence images of dsDNA (green) and mitochondria (red) in the control group and IL-1β treated group. (F) Chondrocytes were pretreated with ethidium bromide 48 h, then 5ng/ml IL-1β with or without TfR1 siRNA was added and western blot was conducted to examine STING, MMP3, MMP13, SOX9 and COL2 proteins expression. The band density was quantified and normalized to control. (G) Representative images of IF staining for STING expression in chondrocytes treated with IL-1β with or without EtBr. Scale bars = 50 μm. Data are presented as mean ± SD from three independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001