Fig 2: Individual role of MAPK signaling in HMGB1-induced RAGE expression in hVSMCs.hVSMCs were transfected with siRNAs for (A) ERK (10 μM), (B) JNK (10 μM), and (C) p38 MAPK (10 μM) for 48 h, and then cells were stimulated with HMGB1 (100 ng/ml) for 24 h. RAGE expression in HMGB1-stimulated cells was determined by Western blot. β-Actin was used as internal controls. Blots are representative of 5 independent experiments. Bottom: Relative intensities of MAPK subfamilies and RAGE to β-Actin were quantified and expressed as the means ± SEMs of 5 independent experiments. MAPK, mitogen-activated protein kinase; HMGB1, high-mobility group box 1; RAGE, receptors for advanced glycation end-product; hVSMCs, human vascular smooth muscle cells. **p < 0.01 vs. corresponding value in negative control.

Fig 3: Role of JNK and Smad3 in TGF‐β1‐induced EMT of BEAS‐2B cells. BEAS‐2B cells were transfected with Smad3‐siRNA (siSmad3), JNK siRNA (siJNK) or siRNA‐scramble (siNC) for 48 h and then exposed to recombinant human TGF‐β1 (10 ng/mL, 48 h) to induced EMT. (A) Representative immunoblots and (B) quantitative analysis of the relative protein levels of Smad3, E‐cadherin, N‐cadherin and Snail in cells; (C) representative immunoblots and (D) quantitative analysis of the relative protein levels of JNK, E‐cadherin, N‐cadherin and Snail in cells; (E) quantitative analysis of the relative protein levels of Smad3 and p‐Smad3 in cells. β‐actin was used as an internal standard. The band intensity was analysed by Bio‐Rad Quantity One v4.62 software. Values are presented as mean ± SEM * P < .05, vs control group (n = 9). # P < .05, vs TGF‐β1 alone group (n = 9)

Fig 4: PP242 induces autophagy by activating JNK.(a) H460 cells were treated with 10 μM PP242 for the indicated amount of time. (b) H460 cells were pretreated with 20 μM SP600125 (JNK inhibitor), 20 μM SB203580 (p38 inhibitor) or 20 μM PD98059 for 1 h prior to treatment with 10 μM PP242 for 12 h. (c) H460 cell were pretreated with 20 μM SP600125 for 1 h and then treated with 10 μM PP242 for 24 h. (d–f) H460 cells were transfected with control or JNK1/2 siRNAs for 16 h, and subsequently treated with 10 μM PP242 for 24 h. (a,c,d,f). The levels of the indicated proteins were estimated using western blot analysis. β-actin was used as a loading control. The blot shown is representative of 2 independent experiments. (b) The percentage of vacuolated cells was calculated from 3 independent images. The data are presented as the mean ± standard deviation (n = 3, ***p < 0.001). (e) Changes in cellular morphology were observed using an inverted microscope (magnification = 400×, scale bar = 5 mm).

Fig 5: Role of ERK and p38 MAPK signaling on HMGB1-augmented proliferation in AGE-stimulated VSMCs.hVSMCs were pre-treated with HMGB1 (100 ng/ml) for 24 h in the presence of various inhibitors for MAPKs including ERK (PD98059, 10 μM), JNK (SP600125, 10 μM), and p38 MAPK (SB203580, 10 μM), and then stimulated with AGE (30 μg/ml) for 24 h. Cell proliferation was determined by an MTT assay. Relative cell proliferation was quantified and expressed as the means ± SEMs of 5 independent experiments. MAPK, mitogen-activated protein kinase; HMGB1, high-mobility group box 1; VSMCs, vascular smooth muscle cells; hVSMCs, human VSMCs. **p < 0.01 vs. corresponding control, #p < 0.05 vs. corresponding value in non-HMGB1, ††p < 0.01 vs. corresponding value in vehicle.