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