Journal: Brain Pathology

Article Title: Neprilysin Protects against Cerebral Amyloid Angiopathy and Aβ‐Induced Degeneration of Cerebrovascular Smooth Muscle Cells

doi: 10.1111/j.1750-3639.2011.00486.x

Figure Lengend Snippet: Aβ toxicity to human cerebrovascular smooth muscle cells (CVSMCs) in vitro. Bar chart showing the percentage cell death at 24 hours caused by adding Aβ in different concentrations to CVSMCs. Error bars indicate the standard error of the mean.

Article Snippet: Culture of CVSMCs Human brain cerebrovascular smooth muscle cells (CVSMCs) were obtained from Sciencell, TCS Cellworks, Bucks, UK.

Techniques: In Vitro

Journal: Brain Pathology

Article Title: Neprilysin Protects against Cerebral Amyloid Angiopathy and Aβ‐Induced Degeneration of Cerebrovascular Smooth Muscle Cells

doi: 10.1111/j.1750-3639.2011.00486.x

Figure Lengend Snippet: NEP protects CVSMCs from Aβ1‐42 cytotoxicity. Reduction in NEP activity was achieved by (A) siRNA knockdown of NEP expression (∼30% reduction in NEP protein, P = 0.007; ∼30% reduction in enzyme activity, P = 0.047) or (B) addition of thiorphan (∼5% reduction in NEP protein, P = 0.046; ∼30% reduction in NEP activity, P = 0.009). Death of CVSMCs on exposure to 10 µM Aβ1‐42 increased ∼20% following siRNA knockdown (P = 0.002) and ∼20% on addition of thiorphan (P = 0.006). Increase in NEP activity was achieved by (C) addition of somatostatin (∼25% increase in NEP protein, P = 0.115; 5% increase in NEP activity, P = 0.730) or (D) transfection of NEP cDNA (∼15% increase in NEP protein, P = 0.046; ∼20% increase in NEP activity, P = 0.026); these manipulations reduced cell death on exposure to 10 µM Aβ1‐42 by ∼15% (P = 0.165) and ∼40% (P = 0.021), respectively. Error bars indicate the standard error of the mean.

Article Snippet: Culture of CVSMCs Human brain cerebrovascular smooth muscle cells (CVSMCs) were obtained from Sciencell, TCS Cellworks, Bucks, UK.

Techniques: Activity Assay, Knockdown, Expressing, Transfection

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 knockdown inhibited the AngII-induced efflux of Cl − in HBVSMCs. a HBVSMCs were treated with angiotensin II (AngII) at different concentrations (10 − 9 , 10 − 8 10 − 7 and 10 − 6 M) for 48 h. Cell viability was determined using the CCK-8 assay. b Intracellular Cl − concentration [Cl − ] i was examined using an MEQ fluorescence probe. c The correlation between [Cl − ] i and cell viability was analyzed. d and e – The expression of ClC-2 in the cells treated as described in ( a ) was examined using western blotting ( d ) and quantitative real-time PCR ( e ). f Cells were treated with ClC-2 siRNA (20 nM) or negative siRNA for 48 h before AngII incubation (10 − 7 M) for a further 48 h. [Cl − ] i was examined. * p < 0.05, ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, n = 6

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: Knockdown, CCK-8 Assay, Concentration Assay, Fluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Control

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 downregulation prevented AngII-induced HBVSMC migration and invasion. a HBVSMCs transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) were subjected to angiotensin II (AngII) treatment (10 − 7 M). The wound healing assay was performed. Representative images are shown (× 100). b The quantification results for the wound closure. c HBVSMC migration was examined via transwell analysis. Representative images are shown (× 100). d The columns represent the relative numbers of invasive cells. ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, n = 6

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: Migration, Transfection, Wound Healing Assay, Control

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 inhibition attenuated the AngII-induced activation of Wnt/β-catenin signaling. a through f HBVSMCs were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) and then stimulated with angiotensin II (AngII; 10 − 7 M) for 48 h. Shown are the western blotting results for β-catenin phosphorylation ( a ), β-catenin cytosol ( b ) and nuclear protein ( c ) levels, GSK-3β phosphorylation ( d ), and survivin ( e ) and cyclin D1 ( f ) protein expression. g Quantitative real-time PCR analysis of Wnt3a and Wnt5a mRNA expression. h The cells were treated with recombinant Wnt3a (100 ng/ml) for 48 h. Wnt3a expression was examined using quantitative real-time. i Viability of HBVSMCs transfected with ClC-2 siRNA followed by co-incubation with recombinant Wnt3a and AngII. ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, $$ p < 0.01 vs. AngII+siClC-2, n = 4

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: Inhibition, Activation Assay, Transfection, Western Blot, Phospho-proteomics, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Incubation, Control

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 knockdown inhibited the AngII-induced efflux of Cl − in HBVSMCs. a HBVSMCs were treated with angiotensin II (AngII) at different concentrations (10 − 9 , 10 − 8 10 − 7 and 10 − 6 M) for 48 h. Cell viability was determined using the CCK-8 assay. b Intracellular Cl − concentration [Cl − ] i was examined using an MEQ fluorescence probe. c The correlation between [Cl − ] i and cell viability was analyzed. d and e – The expression of ClC-2 in the cells treated as described in ( a ) was examined using western blotting ( d ) and quantitative real-time PCR ( e ). f Cells were treated with ClC-2 siRNA (20 nM) or negative siRNA for 48 h before AngII incubation (10 − 7 M) for a further 48 h. [Cl − ] i was examined. * p < 0.05, ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, n = 6

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: CCK-8 Assay, Concentration Assay, Fluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 downregulation prevented AngII-induced HBVSMC migration and invasion. a HBVSMCs transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) were subjected to angiotensin II (AngII) treatment (10 − 7 M). The wound healing assay was performed. Representative images are shown (× 100). b The quantification results for the wound closure. c HBVSMC migration was examined via transwell analysis. Representative images are shown (× 100). d The columns represent the relative numbers of invasive cells. ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, n = 6

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: Migration, Transfection, Wound Healing Assay

Journal: Cellular & Molecular Biology Letters

Article Title: ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/β-catenin signaling pathway

doi: 10.1186/s11658-018-0095-z

Figure Lengend Snippet: ClC-2 inhibition attenuated the AngII-induced activation of Wnt/β-catenin signaling. a through f HBVSMCs were transfected with ClC-2 siRNA (siClC-2; 20 nM) or negative siRNA (negative; 20 nM) and then stimulated with angiotensin II (AngII; 10 − 7 M) for 48 h. Shown are the western blotting results for β-catenin phosphorylation ( a ), β-catenin cytosol ( b ) and nuclear protein ( c ) levels, GSK-3β phosphorylation ( d ), and survivin ( e ) and cyclin D1 ( f ) protein expression. g Quantitative real-time PCR analysis of Wnt3a and Wnt5a mRNA expression. h The cells were treated with recombinant Wnt3a (100 ng/ml) for 48 h. Wnt3a expression was examined using quantitative real-time. i Viability of HBVSMCs transfected with ClC-2 siRNA followed by co-incubation with recombinant Wnt3a and AngII. ** p < 0.01 vs. control, ## p < 0.01 vs. AngII alone, $$ p < 0.01 vs. AngII+siClC-2, n = 4

Article Snippet: Human brain vascular smooth muscle cells (HBVSMCs) were purchased from Creative Bioarray (CSC-7824 W, NY, USA) and cultured in SuperCult Smooth Muscle Cell Medium (Creative Bioarray) containing 10% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified incubator with 5% CO 2 and 95% O 2 at 37 °C.

Techniques: Inhibition, Activation Assay, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Recombinant, Incubation