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Fig. 2 | Arthritis Research & Therapy

Fig. 2

From: IL-1β receptor antagonist (IL-1Ra) combined with autophagy inducer (TAT-Beclin1) is an effective alternative for attenuating extracellular matrix degradation in rat and human osteoarthritis chondrocytes

Fig. 2

IL-1 receptor blockade by IL-1Ra restored autophagy to attenuate ECM degradation in human OA chondrocytes. a Cells were treated with IL-1β (10 ng/ml), IL-1Ra (40 ng/ml), or IL-1β (10 ng/ml)+IL-1Ra (40 ng/ml) for 24 h, respectively. The Col II (1:2000), Aggrecan (1:1000), Beclin1 (1:1000), LC3B (1:1000), and β-actin (1:40,000) protein levels were detected via western blotting. b Cells pretreated with IL-1β (10 ng/ml) for 24 h were treated with IL-1β (10 ng/ml)+IL-1Ra (40 ng/ml) or IL-1β (10 ng/ml)+EBSS (as positive control) for 24 h, respectively. Representative images of the transmission electron microscope (autophagic vacuoles indicated by white arrows). c Cells pretreated with IL-1β (10 ng/ml) for 24 h were treated with IL-1β (10 ng/ml)+IL-1Ra (40 ng/ml) for 24 h. Cells were treated with or without IL-1Ra (40 ng/ml) and IL-1β (10 ng/ml) for 24 h. Representative images of immunofluorescence staining (LC3B (red) (1:400), nucleus staining-DAPI (blue), original magnification× 40). d, e Cells were treated with IL-1β (10 ng/ml), IL-1Ra (40 ng/ml), or IL-1β (10 ng/ml)+IL-1Ra (40 ng/ml) for 24 h, respectively, followed by separation of cytoplasmic and nuclear fractions. The LC3B (1:1000), Lamin B (1:1000), and β-tubulin (1:1000) protein levels were detected via western blotting (d). Protein extracts in nucleus were subjected to immunoprecipitation with anti-LC3B antibody. The immunoprecipitates were immunoblotted with anti-acetylated-lysine antibody (1:1000) (e). f Cells were treated with IL-1β (10 ng/ml), IL-1Ra (40 ng/ml), or IL-1β (10 ng/ml)+IL-1Ra (40 ng/ml) for 24 h, respectively. The mTOR (1:1000), p-mTOR (1:1000), ULK1 (1:1000), p-ULK1 (1:1000), Akt (1:1000), p-Akt (1:1000), NF-κB (p65) (1:1000), and β-actin (1:40,000) protein levels were detected via western blotting. Data is representative of three independent experiments

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