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Figure 4: PPAR regulates LPS-induced release of HMGB1 in RAW 264.7 cells. (a) Cells pretreated for 1 h with GW9662 were stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was collected and subjected to Western blot analysis for determination of HMGB1 levels. (b) Cells transfected with PPAR siRNA for 38 h were incubated with serum-free medium for 24 h, and then treated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis. (c) Cells were transfected with a vector encoding one hairpin siRNA against PPARγ or encoding a scrambled shRNA control. Stable transfectants were selected with 100  g/mL hygromycin, and the expression levels of PPAR were determined by Western blot analysis. (d) Cells expressing PPAR shRNA or scrambled control shRNA were treated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was subjected to Western blot analysis for the determination of HMGB1 levels. (e) Cells transfected with pcDNA3.1-PPAR , or pcDNA3.1 vector for 48 h were harvested and subjected to Western blot analysis with indicated antibodies. (f) Cells transfected with pcDNA 3.1 or pcDNA3.1-PPAR for 48 h were incubated with serum-free medium for 24 h and then stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis for the detection of HMGB1. Ponceau S staining was used as a loading control. The results shown are representative of three independent experiments.