Journal: Frontiers in Pharmacology

Article Title: Bavachin Protects Human Aortic Smooth Muscle Cells Against β -Glycerophosphate-Mediated Vascular Calcification and Apoptosis via Activation of mTOR-Dependent Autophagy and Suppression of β -Catenin Signaling

doi: 10.3389/fphar.2019.01427

Figure Lengend Snippet: Bavachin suppresses β-GP-induced apoptosis in HASMCs. (A) Western blot analysis of apoptosis-related proteins (Caspase3, Caspase9, Bak, Bcl-2, and Bax) in bavachin and β-GP-treated HASMCs ( * P < 0.05 , ** P < 0.01 , *** P < 0.001 ). The full-length images of Western blot are shown in Figure S4A . (B) Flow cytometry analysis of apoptosis in bavachin and β-GP-treated HASMCs ( *** P < 0.001 ). Statistical significance was analyzed by the one-way ANOVA (* P < 0.05 , ** P < 0.01 , *** P < 0.001 ). The data is represented as mean ± S.D. (n = 3).

Article Snippet: For the detection of cytotoxicity, cells were stained with propidium iodide (50 µg/ml) and annexin V-FITC (2.5 µg/ml) at RT for 15 min with FITC Annexin V Apoptosis Detection Kit (BD, USA) in the dark as described previously ( ).

Techniques: Western Blot, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Bavachin Protects Human Aortic Smooth Muscle Cells Against β -Glycerophosphate-Mediated Vascular Calcification and Apoptosis via Activation of mTOR-Dependent Autophagy and Suppression of β -Catenin Signaling

doi: 10.3389/fphar.2019.01427

Figure Lengend Snippet: Bavachin suppresses β-GP-induced calcification and apoptosis via induction of autophagy. Effects of autophagy inhibitor WM in β-GP/bavachin-treated HASMCs. (A) A representative plate view of VONKOSSA staining was shown after application of WM in β-GP/bavachin-treated HASMCs. The black spots were observed in cytoplasm and nuclear regions of cells. (B) Immunofluorescence staining of calcification and autophagy-related proteins. Green GFP signal represents the calcified protein OPN, whereas red TRITC signal represents the autophagy protein LC3, and blue DAPI signal represents the nuclear region (scalebar = 25 μm). (C) Western blot analysis of autophagy proteins (LC3 and beclin1) in HASMCs treated with 10 mM β-GP and 25 μM bavachin for 72 h (** P < 0.01 , *** P < 0.001 ). The full-length images of Western blot are shown in Figure S7C . (D) Western blot analysis of apoptotic proteins (Caspase3, Caspase9, Bak, Bcl-2, and Bax). The full-length images of Western blot are shown in Figures S1A, S7D . (E) Western blot analysis of calcification-related proteins: Wnt3a, β-catenin, OPN, BMP2, and Runx2. The full-length images of Western blot are shown in Figures 1B , S7E . Statistical significance was analyzed by the one-way ANOVA (** P < 0.01 , *** P < 0.001 ). The data is represented as mean ± S.D. (n = 3).

Article Snippet: For the detection of cytotoxicity, cells were stained with propidium iodide (50 µg/ml) and annexin V-FITC (2.5 µg/ml) at RT for 15 min with FITC Annexin V Apoptosis Detection Kit (BD, USA) in the dark as described previously ( ).

Techniques: Staining, Immunofluorescence, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Bavachin Protects Human Aortic Smooth Muscle Cells Against β -Glycerophosphate-Mediated Vascular Calcification and Apoptosis via Activation of mTOR-Dependent Autophagy and Suppression of β -Catenin Signaling

doi: 10.3389/fphar.2019.01427

Figure Lengend Snippet: Bavachin inhibits calcification and cell apoptosis in β-GP-treated HASMCs via Atg7-dependent autophagy. (A) Protein expression difference in Atg7 knockdown HASMCs. Western blot analysis of apoptosis-related proteins (Bax) and calcification-related protein (Runx2 and BMP2) in β-GP treated HASMCs transfected with or without Atg7 siRNA. (* P < 0.05 , ** P < 0.01 , *** P < 0.001 ). The full-length images of Western blot are shown in Figure S8A . (B) Western blot analysis of calcification-related proteins (β-catenin, Runx2, and OPN) in bavachin and β-GP-treated HASMCs transfected with or without Atg7 siRNA. The full-length images of Western blot are shown in Figure S8B . (C) Western blot analysis of apoptosis-related proteins (Bax and Bak) and LC3-II conversion in bavachin and β-GP-treated HASMCs transfected with or without Atg7 siRNA. The full-length images of Western blot are shown in Figure S8C . Statistical significance was analyzed by the one-way ANOVA (* P < 0.05 , ** P < 0.01 , *** P < 0.001 ). The data is represented as mean ± S.D. (n = 3).

Article Snippet: For the detection of cytotoxicity, cells were stained with propidium iodide (50 µg/ml) and annexin V-FITC (2.5 µg/ml) at RT for 15 min with FITC Annexin V Apoptosis Detection Kit (BD, USA) in the dark as described previously ( ).

Techniques: Expressing, Western Blot, Transfection

Journal: Frontiers in Pharmacology

Article Title: Bavachin Protects Human Aortic Smooth Muscle Cells Against β -Glycerophosphate-Mediated Vascular Calcification and Apoptosis via Activation of mTOR-Dependent Autophagy and Suppression of β -Catenin Signaling

doi: 10.3389/fphar.2019.01427

Figure Lengend Snippet: The possible mechanism by which Bavachin protects HASMCs against β-glycerophosphate-mediated VC and apoptosis, occurs via the activation of mTOR-dependent autophagy and suppression of β-catenin signalling.

Article Snippet: For the detection of cytotoxicity, cells were stained with propidium iodide (50 µg/ml) and annexin V-FITC (2.5 µg/ml) at RT for 15 min with FITC Annexin V Apoptosis Detection Kit (BD, USA) in the dark as described previously ( ).

Techniques: Activation Assay