Hypoxia Inhibits Osteogenesis and Promotes Adipogenesis of Fibroblast-like Synoviocytes via Upregulation of Leptin in Patients with Rheumatoid Arthritis

Figure 4

Knockdown of leptin reversed the hypoxia-mediated reduction of osteogenesis of RA-FLSs under hypoxia condition. (a) The knockdown efficacy of siRNAs targeting leptin evaluated by RT-qPCR. NC: Negative Control; siLep: siRNA-leptin. Data quantification is (-test, three independent experiments). (b) The absence of leptin in RA-FLSs was analyzed by Western blot. Data quantification is (-test, three independent experiments). (c) RT-qPCR analysis of leptin and osteogenesis-related genes (Runx2, Ocn, and Alp) in leptin-knockdown or scrambled siRNA control RA-FLSs after culture with osteogenic induction medium for 3 days under hypoxia and normoxia condition. Data quantification is (-test, three independent experiments). (d, e) Westernblot shows leptin expression and osteogenesis-related proteins (RUNX2, OSX, and OCN) in leptin-knockdown or scramble siRNA control RA-FLSs after culture with osteogenic induction medium for 3 days under hypoxia and normoxia condition. The expression normalized with β-actin. Data quantification is (-test, three independent experiments). (f) Alizarin Red staining of the leptin-knockdown or scrambled siRNA control RA-FLSs after culture with osteogenic induction for 21 days under hypoxia and normoxia conditions. (g) Alizarin Red positive cells per field were counted. Data quantification is . (-test, six randomized fields, three independent experiments, and one representative experiment shown). ; ; ; . Scale bars: .

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