KLF2 Protects against Osteoarthritis by Repressing Oxidative Response through Activation of Nrf2/ARE Signaling In Vitro and In Vivo

Figure 4

KLF2 exerts chondroprotective effects through the induction of Nrf2 nuclear translocation. (a, b) SW1353 cells were treated with ML385 (an inhibitor of Nrf2). After 12 h, Nrf2 expression was evaluated by real-time PCR (a) and Western blotting analysis (b). β-Actin was used as an endogenous control. Quantitative analysis of Nrf2 protein levels based on specific signal intensities measured using Image. (c–e) SW1353 cells were infected with nonspecific control virus (Lv-NC) or a KLF2-expressing virus (Lv-KLF2) or incubated with the pharmacological activator of KLF2 simvastatin (Sim). After 24 h, cells were treated with or without ML385 for 12 h. HO-1 and NQO1 mRNA levels were evaluated by real-time PCR (c), and protein levels were evaluated by Western blot analysis (d). β-Actin was used as an endogenous control. Quantitative analysis of HO-1 and NQO1 protein levels based on specific signal intensities measured using ImageJ (e). (f, g) SW1353 cells were infected with Lv-NC or Lv-KLF2. Nrf2 protein levels in the cytosolic and nuclear fractions were evaluated by Western blotting analysis (f). α-Tubulin (cytosolic) and Lamin B (nuclear) were used as endogenous controls. Quantitative analysis of Nrf2 protein levels based on specific signal intensities measured using ImageJ. (g) Immunohistochemical staining of the subcellular localization of Nrf2 protein (red). Nuclei were stained with DAPI (blue). The scale bars represent 10 μm. (h, i) SW1353 cells were infected with Lv-NC or Lv-KLF2 or incubated with simvastatin. After 24 h, cells were treated with or without ML385 for 12 h prior to 24 h of treatment with 20 ng/ml IL-1β. MMP13 mRNA levels were evaluated by real-time PCR (h), and apoptosis was determined by Annexin V/PI staining followed by flow cytometry (i). All data are expressed as the . and .

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