Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

doi: 10.3389/fimmu.2021.641973

Figure Lengend Snippet: The expression of TGF-β1 in cardiac allografts. Cardiac tissue samples were collected at indicated time after transplantation. (A) Expression of TGF-β1 protein in allografts. (B) The relative quantity of TGF-β1 protein normalized to GAPDH. The data of (B) was shown as mean ± SEM (n=3/group). *: p <0.05; **: p <0.01, ***: p <0.001. (C) The localization of TGF-β1 in allografts was examined by immunohistochemistry staining. TGF-β1 was stained brown and the nuclei was counterstained blue. Upper image: scale bar: 50 μm; Lower image: enlarged view of the box above. The data were representative of three independent experiments.

Article Snippet: Total proteins were extracted from cardiac grafts or cell lysates after recombinant HMGB1 (rHMGB1) (Sigma-Aldrich, Saint Louis, MO, USA) stimulation, and subjected to immunoblots as well as incubation with mouse monoclonal anti-TGF-β1 (1:500, Abcam plc, Cambridge, UK), rabbit monoclonal anti-α-SMA (1:10,000, Abcam plc, Cambridge, UK), rabbit polyclonal anti-p-Smad2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, USA), rabbit polyclonal anti-p-Smad3 (1:500, Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad2 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad3 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) or rabbit polyclonal anti-GAPDH (1:1,000, ZSGQ-BIO, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Transplantation Assay, Immunohistochemistry, Staining

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

doi: 10.3389/fimmu.2021.641973

Figure Lengend Snippet: HMGB1 blockade attenuated the synthesis and release of TGF-β1 in allografts. (A) Representative images showed the protein expression of TGF-β1 (brown arrows) by immunohistochemical analysis in cardiac allografts at week 8 post transplantation in two groups. Scale bar: 50 μm. (B) Concentration of TGF-β1 in recipients' sera and the allograft homogenate at week 8 post-transplant tested by ELISA. Values were expressed as means ± SEM ( n = 4–8/group). **: p <0.01; ***: p <0.001, vs. the IgG group. (C) The protein expression of TGF-β1 by western blotting after HMGB1 mAb administration. (D) The relative quantity of TGF-β1 protein normalized to GAPDH. Data were shown as mean ± SEM. ***: p <0.001, vs. the IgG group. All data were representative of three independent experiments.

Article Snippet: Total proteins were extracted from cardiac grafts or cell lysates after recombinant HMGB1 (rHMGB1) (Sigma-Aldrich, Saint Louis, MO, USA) stimulation, and subjected to immunoblots as well as incubation with mouse monoclonal anti-TGF-β1 (1:500, Abcam plc, Cambridge, UK), rabbit monoclonal anti-α-SMA (1:10,000, Abcam plc, Cambridge, UK), rabbit polyclonal anti-p-Smad2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, USA), rabbit polyclonal anti-p-Smad3 (1:500, Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad2 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad3 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) or rabbit polyclonal anti-GAPDH (1:1,000, ZSGQ-BIO, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Immunohistochemical staining, Transplantation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

doi: 10.3389/fimmu.2021.641973

Figure Lengend Snippet: Extracellular HMGB1 promoted the release of TGF-β1 from fibroblasts. (A) The expression of TGF-β1 on myofibroblasts (α-SMA+) in the allograft at week 8 posttransplant and HMGB1 mAb treatment. α-SMA was labeled with green fluorescence, TGF-β1 was labeled with red color and nuclei were stained with DAPI in blue. Scale bar: 25 μm. (B) The protein expression of α-SMA by western blotting after HMGB1 mAb administration and (C) the relative quantity of α-SMA protein normalized to GAPDH in grafts. After 16 h of serum starvation, stimulation with rHMGB1 for 48 h at dose of 0 or 500 ng/ml in media containing 0.5% FBS, (D) western blotting analysis of α-SMA protein expression in cultured cardiac fibroblasts, (E) the relative quantity of α-SMA protein normalized to GAPDH, (F) free active and total TGF-β1 in the supernatants of fibroblasts after rHMGB1 stimulation detected by ELISA. Data were expressed as mean ± SEM (n=3/group) and representative of at least three separate experiments. *: p <0.05; **: p <0.01; ***: p <0.001, vs. negative control.

Article Snippet: Total proteins were extracted from cardiac grafts or cell lysates after recombinant HMGB1 (rHMGB1) (Sigma-Aldrich, Saint Louis, MO, USA) stimulation, and subjected to immunoblots as well as incubation with mouse monoclonal anti-TGF-β1 (1:500, Abcam plc, Cambridge, UK), rabbit monoclonal anti-α-SMA (1:10,000, Abcam plc, Cambridge, UK), rabbit polyclonal anti-p-Smad2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, USA), rabbit polyclonal anti-p-Smad3 (1:500, Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad2 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad3 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) or rabbit polyclonal anti-GAPDH (1:1,000, ZSGQ-BIO, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Labeling, Fluorescence, Staining, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

doi: 10.3389/fimmu.2021.641973

Figure Lengend Snippet: Extracellular HMGB1 enhanced macrophage released TGF-β1. (A) The expression of TGF-β1 on macrophages (F4/80+) in the allograft at week 8 posttransplant and HMGB1 mAb treatment. F4/80 was labeled with red color, TGF-β1 was labeled with green fluorescence and nuclei were stained with DAPI in blue. Scale bar: 25 μm. (B) After 16 h of serum starvation, BMMs were treated with recombinant HMGB1 at 500 ng/mL for 48h in the presence of 0.5% FBS. Free active and total TGF-β1 in the supernatants of BMMs were detected by ELISA. Data were expressed as means ± SEM ( n = 3/group). **: p <0.01; ***: p <0.001, vs. negative control. All data were representative of three independent experiments.

Article Snippet: Total proteins were extracted from cardiac grafts or cell lysates after recombinant HMGB1 (rHMGB1) (Sigma-Aldrich, Saint Louis, MO, USA) stimulation, and subjected to immunoblots as well as incubation with mouse monoclonal anti-TGF-β1 (1:500, Abcam plc, Cambridge, UK), rabbit monoclonal anti-α-SMA (1:10,000, Abcam plc, Cambridge, UK), rabbit polyclonal anti-p-Smad2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, USA), rabbit polyclonal anti-p-Smad3 (1:500, Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad2 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad3 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) or rabbit polyclonal anti-GAPDH (1:1,000, ZSGQ-BIO, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Labeling, Fluorescence, Staining, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: Extracellular HMGB1 Contributes to the Chronic Cardiac Allograft Vasculopathy/Fibrosis by Modulating TGF-β1 Signaling

doi: 10.3389/fimmu.2021.641973

Figure Lengend Snippet: HMGB1 induced phosphorylation of Smad2 and Smad3 in macrophages and fibroblasts. (A,C) The protein expression of p-Smad2 and p-Smad3 by western blot after HMGB1 Ab administration. (B,D) The relative quantity of p-Smad2, p-Smad3 protein normalized to total Smad2 and Smad3. Data were shown as mean ± SEM and represented of at least three separate experiments. *: p <0.05; **: p <0.01, ***: p <0.001, vs. the IgG group. All data were representative of three independent experiments. (E–H) After 16 h of serum starvation, cultured macrophages and fibroblasts were treated with rHMGB1 for 0, 10, 30 and 60 min in the presence of 0.5% FBS. The phosphorylation of Smad2 and Smad3 in BMMs (E,F) and fibroblasts (G,H) was observed with western blot. The relative quantity of p-Smad2 and p-Smad3 normalized to total Smad2 and Smad3, respectively. The data were shown as mean ± SEM ( n = 3/group). **: p <0.01; ***: p <0.001, vs. negative control. All data were representative of three independent experiments. (I,J) ELISA to detect for free active and total TGF-β1 levels of BMMs (I) and fibroblasts (J) cultured supernatant. The data were shown as mean ± SEM ( n = 3/group). *: p <0.05; **: p <0.01; ***: p <0.001, vs. negative control. All data were representative of two independent experiments.

Article Snippet: Total proteins were extracted from cardiac grafts or cell lysates after recombinant HMGB1 (rHMGB1) (Sigma-Aldrich, Saint Louis, MO, USA) stimulation, and subjected to immunoblots as well as incubation with mouse monoclonal anti-TGF-β1 (1:500, Abcam plc, Cambridge, UK), rabbit monoclonal anti-α-SMA (1:10,000, Abcam plc, Cambridge, UK), rabbit polyclonal anti-p-Smad2 (1:200, Santa Cruz Biotechnology, Inc., Dallas, USA), rabbit polyclonal anti-p-Smad3 (1:500, Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad2 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China), rabbit polyclonal anti-Smad3 (1:500, Shanghai Sangon Biotechnology Co., Ltd., Shanghai, China) or rabbit polyclonal anti-GAPDH (1:1,000, ZSGQ-BIO, Beijing Zhong Shan Jin Qiao Biotechnology Co., Ltd., Beijing, China).

Techniques: Expressing, Western Blot, Cell Culture, Negative Control, Enzyme-linked Immunosorbent Assay