Fig 2: Systemic IL-10 induced HO-1 expression in the infarcted hearts in diabetic mice.Diabetic mouse left ventricular tissues on day 3 after MI, including the infarct and border zone, were lysed and measured for HO-1 protein expression by Western blot. GAPDH was used as a loading control. (A) Representative blots of HO-1 and GAPDH. (B) Quantitative analysis of HO-1 expression in diabetic hearts after MI. One-way ANOVA followed by Tukey’s test. n = 3 per group, *P < 0.05 vs. sham. (C) Left: immunofluorescence staining of HO-1 (green fluorescence) in the border zones of heart sections on day 3 after MI in control and diabetic mice. Blue color is the DAPI staining. Scale bar: 50 μm. Right: quantitative data of HO-1+ cells on day 3 after MI for control and diabetic mouse hearts. Independent t test. n = 9, *P < 0.05 vs. Con MI.

Fig 3: Co-expression of HER2CA and FLAG-NRF2 induces the expressions of NRF2-target proteins and increased the NRF2-dependent transcription in MCF7 cells.(A) Overexpression of HER2CA increases the NRF2-induced expressions of HO-1 and MRP5 in MCF7 cells. MCF7 cells were transiently transfected with expression vectors by Lipofectamine PLUS as indicated and subjected to western blot analysis. β-actin was used as a loading control. The blots used in this experiment was cut into several pieces according to estimated M.W. of proteins of interest and probed indicated antibodies. Different pieces of the same protein blots or same samples with same conditions of electrophoresis and electrotransfer were used and presented. (B) HER2CA increases NRF2 DNA-binding in MCF7 cells. MCF7 cells were transfected with expression vectors as indicated and NRF2 DNA-binding assay was performed. As a control, MCF7 cells were treated with 100 µM of tBHQ for 8 hr. (C) HER2CA increases the ARE-Luc reporter gene activity in an NRF2-depedent manner. MCF7 cells were transfected with expression vectors as indicated with Lipofectamine PLUS and luciferase activity was determined as described in Materials and methods. (D) HER2CA increased transcriptional activation of HO-1 promoter by endogenous NRF2. MCF7 cells were transfected with HO-1 promoter-Luc and increasing amounts of HER2CA expression vector in the absence or presence of KEAP1 knockdown. Luciferase assay were performed as described in (C). (E) HER2CA increases the NRF2-dependent transcription of HO-1 promoter. MCF7 cells transfected with expression vectors by Lipofectamine PLUS as indicated in the presence of HO-1 promoter-Luc containing either wild type or mutant AP-1 site. Luciferase activity was determined as in (B). (B,C) Representative data were presented as mean ± SEM of triplicate experiments. Statistically significance was determined by two-tailed Student's t-test. *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.

Fig 4: Cinnamic aldehyde (CA) activated the Nrf2 pathway in ZDF vascular smooth muscle cells (VSMC). (A-C) ZDF VSMC in 25 mM glucose were synchronized overnight and then stimulated with 25 ng/mL PDGF-BB with or without varying concentrations of CA. (A) Immunoblot of nuclear and cytoplasmic ZDF VSMC fractions treated with 100 µM CA or 4 µM sulforaphane (SFN) for 6 h probed for Nrf2 with TATA Binding Protein (TATA BP) or β-Actin in the nuclear and cytoplasmic fraction respectively. Plot of nuclear fraction Nrf2 band density normalized to TATA BP. (B) Confocal images of subcellular Nrf2 localization after CA or SFN treatment for 24 h. Green = Nrf2; Blue = Nuclei. (C) Representative immunoblot of whole cell lysates treated with CA or SFN for 4 h probed for heme oxygenase 1 (HO-1). Plot of HO-1 band density normalized to β-Actin (n = 3). (D) Hyperglycemic ZDF rats weighing 350–500 g underwent the carotid balloon injury and were treated with periadventitial application of Pluronic vehicle (100 µL) or CA in Pluronic (100 µM in 100 µL) at the time of surgery. Carotids were harvested three days after surgery. Confocal images of three-day injured carotid arteries stained for Nrf2. Red = Nrf2; Blue = DAPI; Green = elastic lamina; L = lumen. Arrows indicate nuclear co-localization. *p < 0.05 compared to control (Ctl); one-way ANOVA with Tukey's correction. Data presented as means ± SEM (n = 3 independent experiments in triplicate; n = 3 ZDF VSMC or tissue cross-sections for confocal images with representative image shown).

Fig 5: IL-10 induced CD163 and HO-1 expression in THP-1 macrophages cultured under hyperglycemia.THP-1 cells were differentiated to macrophages and cultured under high glucose conditions (25 mM). (A and B) Cells were treated as described in Methods, and CD163 (A) and HO-1 (B) protein expression were measured by Western blots. GAPDH was used as a loading control. Top: representative blots. Bottom: quantitative data. One-way ANOVA, followed by Tukey’s test; n = 3 per group, *P < 0.05 vs. control; #P < 0.05 vs. Hb; &P < 0.05 vs. IL10.