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

Journal: Frontiers in Pharmacology

Article Title: Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

doi: 10.3389/fphar.2013.00014

Figure Lengend Snippet: Time-course of 5-HT uptake in HBVSMCs . [ 3 H]5-HT uptake (1 μM, 2 μCi/mL) was measured in HBVSMCs in the presence or absence of Na + as indicated. Values are means ± SEM of three experiments carried out in triplicate.

Article Snippet: Primary culture of HBVSMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in the medium supplied by the same company, at 37°C in an atmosphere of 95% air and 5% CO 2 .

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

doi: 10.3389/fphar.2013.00014

Figure Lengend Snippet: Kinetic analyses of 5-HT uptake in HBVSMCs . Concentration dependence of 5-HT (0.1 μM to 50 mM) uptake was determined by measuring [ 3 H]5-HT uptake at room temperature for 30 min. Values are means ± SEM of three experiments carried out in triplicate.

Article Snippet: Primary culture of HBVSMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in the medium supplied by the same company, at 37°C in an atmosphere of 95% air and 5% CO 2 .

Techniques: Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

doi: 10.3389/fphar.2013.00014

Figure Lengend Snippet: Effects of various transporter inhibitors on 5-HT uptake in HBVSMCs . [ 3 H]5-HT uptake (1 μM, 2 μCi/mL) was measured at room temperature for 30 min in the presence of various concentrations of citalopram (■), desipramine (□), GBR12935 (●), and corticosterone (◯). Values are means ± SEM of three experiments carried out in triplicate.

Article Snippet: Primary culture of HBVSMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in the medium supplied by the same company, at 37°C in an atmosphere of 95% air and 5% CO 2 .

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

doi: 10.3389/fphar.2013.00014

Figure Lengend Snippet: Reverse transcription-polymerase chain reaction analyses of various 5-HT transporter mRNAs in HBVSMCs . PCR products are seen in reactions using oligonucleotide primer pairs for (A) OCT-3 and (B) PMAT but not for (A) OCT-1, OCT-2, (C) SERT, (D) NET, and (E) DAT. Positive controls with human liver or brain cDNA indicate the expected sizes of amplified fragments: 363 bp (OCT-1), 334 bp (OCT-2), 419 bp (OCT-3), 319 bp (SERT), 400 bp (PMAT), 395 bp (NET), and 370 bp (DAT).

Article Snippet: Primary culture of HBVSMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in the medium supplied by the same company, at 37°C in an atmosphere of 95% air and 5% CO 2 .

Techniques: Reverse Transcription, Polymerase Chain Reaction, Amplification

Journal: Frontiers in Pharmacology

Article Title: Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

doi: 10.3389/fphar.2013.00014

Figure Lengend Snippet: Effects of siRNA knockdown of OCT-3 and PMAT on 5-HT uptake in HBVSMCs . (A) mRNA and (B) protein expressions of OCT-3 and PMAT mRNA in HBVSMCs transfected with siRNA against OCT-3 and PMAT and a control non-silencing sequence. Bar graph showing the amount of (C) mRNA and (D) protein of OCT-3 and PMAT normalized to β-actin. (E) 5-HT uptake in HBVSMCs transfected with OCT-3 and PMAT siRNA and a control non-silencing sequence. Values are means ± SEM of three separate experiments. * P < 0.05 vs. control.

Article Snippet: Primary culture of HBVSMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in the medium supplied by the same company, at 37°C in an atmosphere of 95% air and 5% CO 2 .

Techniques: Knockdown, Transfection, Control, Sequencing