Figure 3

LIPUS up‐regulates chondrocyte autophagy in a calcium pathway‐dependent manner. A) Schematic of the mouse experiment procedures. B) Immunohistochemical staining of LC3 and ATG7 in the articular cartilage. Scale bar, 50 µm. Quantification of LC3‐positive cells C) and ATG7‐positive cells D) (n = 3 mice). E) Schematic of the inflammatory chondrocyte establishment and treating with LIPUS. F) The chondrocytes we have obtained observing by microscope. Scale bar, 20µm. G) TEM was used for detection the autophagosomes in chondrocytes following LIPUS treatment. Red arrowhead indicates an autophagosome. Scale bar, 1 µm. H) Observation the autophagy flux of living chondrocyte labeled by DAPRed. I) Autophagy flux was dynamically observed by DAPRed at different time point after LIPUS. White arrowhead indicates the significant changes. Scale bar, 10 µm. WB (J) and densitometry analysis (K) of CAMK II protein expression following LIPUS treatment (n = 3). WB L) and densitometry analysis M) of AMPK protein expression following LIPUS treatment at different time point (n = 3). N) Immunofluorescence was used to detect the LC3‐puncta after the chondrocytes was co‐incubated with BAPTA‐AM. Scale bar, 5 µm. O) Quantification of LC3‐puncta in chondrocytes (n = 4). Data are presented as means ± SD. Statistical analysis was performed using Student's t test. ****(P < 0.0001), ***(P < 0.001), **(P < 0.01), *(P < 0.05), ns (0.05 < P).