Fig 2: Identification of the transcription factors mediating OPN expression in HMGB1-stimulated VSMCs. (A) VSMCs were transfected with various promoter constructs or an empty luciferase vector (pGL3) for 48h and then stimulated with HMGB1 (100ng/ml) for 3h. Relative luciferase activities were expressed as the mean±SEM of four independent experiments. **p<0.01 vs. corresponding value in control. (B) Nucleotide sequence of the –538~–234 promoter region of the OPN gene is shown. The sequence of potential binding sites for AP-1 and C/EBPβ in pLuc-OPN-538 were underlined. The transcription factor-binding sites were identified using TF search software. (C) VSMCs were transfected with AP-1 and C/EBPβ siRNAs (200nM) for 48h and then stimulated with HMGB1 (100ng/ml) for 48h. The expression of OPN was determined by Western blot. β-actin was used as a control. Relative intensities were expressed as the mean±SEM of five independent experiments. **p<0.01 vs. corresponding value in negative control in vehicle and ##p<0.01 vs. value in negative control in HMGB1.

Fig 3: The role of OPN on HMGB1-induced VSMC migration. (A) VSMCs were pretreated with MPIIIB10 (30 to 300ng/ml) or IgG (1μg/ml) for 1h and then stimulated with HMGB1 (100ng/ml) for 48h. Cell migration was determined by wound-healing assay. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μm. Data were quantified, expressed as the mean±SEM of five independent experiments. **p<0.01 vs. value in control and #p<0.05 vs. corresponding value in vehicle. (B) VSMCs were transfected with negative control siRNA or OPN siRNA (200nM) for 48h and then stimulated with HMGB1 (100ng/ml) for 48h. Cell migration was determined by wound-healing assay. Cell migration area (%) was calculated from the ratio of changes in wound area. Scale bar: 500μm. Data were quantified, expressed as the mean±SEM of four independent experiments. **p<0.01 vs. value in negative control in vehicle and #p<0.05 vs. corresponding value in negative control in HMGB1.

Fig 4: HMGB1 enhances M1-like polarization of macrophages by activating ERK1/2-NFκB-NLRP3 inflammasome pathway. A Cytokines released into supernatants of THP1 cell line -derived macrophages, including IFN-γ, IL-1β, IL-6, IL-8, CCL2 and TNF-α were detected by ELISA after indicated treatment for 24 h. rhHMGB1: 1 μg/ml; FBS-ZM1: 100 nM; CY-09: 5 μM. B Immunoblot of p-ERK1/2, p-IKB and p-NFκB in lysates of THP1 cell line-derived macrophages treated with rhHMGB1 (100 ng/ml) for indicated times. C THP1 cell line-derived macrophages were pre-treated with FBS-ZM1 (100 nM) for 2 h, then stimulated with rhHMGB1 (1 μg/ml) for 24 h. p-ERK1/2, p-IKB, p-NFκB, NLRP3 and ASC levels were detected by immunoblot. D Representative IF of the cytokines (M1-like: TNF-α, IFN-γ, IL-1β, IL-6) (red) and HMGB1 (green) in GL261 cell-derived xenograft tumors treated with TMZ plus rmHMGB1. Squares are enlarged and shown on the right side of each image. Scale bars = 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance

Fig 5: RAGE blockade inhibits HMGB1-induced hair shaft elongation in human hair follicles. Human hair follicles were pre-treated with 10 μg/ml RAGE-FC for 1 h followed by 200 ng/ml HMGB1 for 9 days. (a) Representative photos of hair follicles from each group are shown. (b) Quantification of hair shaft growth rates. The changes in hair shaft lengths were quantified at 6 and 9 days using a stereomicroscope. Data are shown as the mean ± SD of five independent donors (at least 120 follicles for each experiment. p-values were determined by ANOVA followed by Bonferroni test. **p < 0.01 and ***p < 0.001 compared with control group. (c) Human hair follicles were pre-treated with 10 μg/ml RAGE-FC for 1 h followed by 200 ng/ml HMGB1 for 3 days. Representative confocal images of hair follicles stained with COX-1 (red) and mPGES-1 (green) are shown. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. On day 3, each hair follicle was frozen and cryosectioned (6 μm). Data are representative of three independent experiments. Scale bar = 50 μm.