Fig. 6.

Calcium influx was required for mechanical stimulus-induced ferroptosis in chondrocytes. (A). Calcium influx in chromocytes was tested by Fluo-4 AM of chondrocytes in each indicated group. Scale bars 50 μm. (B). Quantitative analysis of fluorescence intensity (n = 3 for each group). (C). Cell death ratio of chondrocytes in each indicated group. Scale bar = 50 μm. (D). The cell number of PI (red fluorescence)/calcein (green fluorescence) reflected the cell death ratio (n = 3 for each group). (E). The expression of GSH in chondrocytes (n = 3 for each group). (F). Representative images of ROS levels in chondrocytes in each indicated group. Scale bar = 50 μm. (G). Quantitative analysis of fluorescence intensity (n = 3 for each group). (H). Representative fluorescence images of mitochondria in chondrocytes. Scale bar = 50 μm. (I). Quantitative analysis of fluorescence intensity (n = 3 for each group). (J). JC-1 assay of chondrocytes in each indicated group. Scale bar = 50 μm. (K). The relative IOD ratio of red fluorescence to green fluorescence was used for quantitative analysis (n = 3 for each group). (L). Real-time PCR analysis of GPX4, Aggrecan, Col2, ADAMTS-5 and MMP-13 in chondrocytes (n = 3 for each group). (M). Representative immunofluorescence images of GPX4, Col2 and ADAMTS-5 in chondrocytes. Scale bars 20 μm. (N-P). Quantification of immunofluorescence analysis (n = 3 for each group). (Q). Western blot (WB) analysis of GPX4, Col2, ADAMTS-5 and MMP-13 in chondrocytes. (R). Quantification of WB analysis (n = 3 for each group). (S). Schematic diagram of mechanical overloading induced ferroptosis in chondrocyte. Data were presented as the mean ± SD. *P < 0.05, **P < 0.01.