Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Impaired BK-β1-mediated channel activation, reduced BK-β1 expression, and increased MuRF1 expression in diabetic vessels and in HG-cultured human coronary SMCs. A, inside-out single-channel BK currents were elicited at +60 mV in freshly isolated coronary SMCs from control and diabetic mice before and after exposure to DHS-1 (0.1 μm). DHS-1 robustly increased BK channel NPo in control SMCs, but its effects were diminished in diabetic SMCs. Dashed lines represent the channel closed state (c). B, left panel, the cell lysates of control and diabetic mouse aortas were blotted against anti-ubiquitin antibody. Right panel, the immunoprecipitates (IPs) of anti-BK-β1 antibodies were blotted against anti-ubiquitin (Ub) antibody, showing a significant increase of ubiquitinated BK-β1 protein in diabetic mice. Ctrl, control. C and D, a marked reduction of BK-β1 expression in the aortas of diabetic mice compared with those of control mice and in human coronary SMCs after a 2-week culture with 22 mm glucose (HG) compared with that with 5 mm glucose (NG) culture. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Activation Assay, Expressing, Cell Culture, Isolation, Control, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of BK-β1 expression by MuRF1. A, after a 2-week culture in NG or HG, BK-β1 protein expression in human coronary SMCs was determined under the following conditions: NG culture alone, HG culture after a 48-h transfection with control siRNA (0.1 μm), and HG culture after a 48-h transfection with MuRF1 siRNA (0.1 μm). MuRF1 siRNA suppressed MuRF1 expression under HG culture conditions to that of NG culture conditions. Ctrl, control. B, FLAG-BK-β1 or FLAG-BK-α stably expressed in HEK293 cells 48 h after cotransfection with Myc-MuRF1 WT cDNA or Myc-MuRF1ΔR mutant cDNA. The BK-β1 protein level was halved in cells with MuRF1 WT transfection, but BK-α expression remained unchanged. N.S., not significant. C and D, coimmunoprecipitation experiment shows attenuated BK-β1 protein ubiquitination in the pulldown complexes with anti-HA-ubiquitin (anti-HA-Ub) antibodies or with anti-FLAG antibodies in HEK293 cells after a 24-h transfection with MuRF1ΔR compared with those with MuRF1 WT. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Transfection, Control, Stable Transfection, Cotransfection, Mutagenesis, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Reduced BK-β1 expression and BK-β1-mediated channel activation in vascular SMCs by MuRF1 expression. A, fluorescence microscopy images illustrate GPF expression in endothelially denudated mouse coronary artery 12 h after transduction with Ad-GFP but not in a negative control artery. Immunoblot analyses show increased MuRF1 and decreased BK-β1 expression in the aortas of control (Ctrl) mice after a 12-h transduction with Ad-MuRF1 compared with controls with Ad-GFP transduction. B, inside-out single BK currents were elicited from +60 mV at baseline, after exposure to 0.1 μm DHS-1, and after washout (W/O). DHS-1 had no effect on mouse coronary SMCs 12 h after transduction with Ad-MuRF1. Dashed lines represent the channel closed state (c). Group data with statistical analysis are shown in the bar graphs. N.S., not significant. C, effects of Ad-MuRF1 transduction on dose-dependent, NS1619-mediated (10−9-10−5 m) dilation of endothelium-denuded coronary arteries from non-diabetic mice. Transduction with Ad-MuRF1 (12 h) attenuated the NS1619-induced vasodilation, but the NS1619 response was preserved in Ad-MuRF1-treated coronaries by incubation with MG132 (10 μm, 12 h). Control vessels received Ad-GFP transduction. *, p < 0.05, Ad-MuRF1 versus Ad-GFP; +, p < 0.05 Ad-MuRF1+MG132 versus Ad-GFP.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Fluorescence, Microscopy, Transduction, Negative Control, Western Blot, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Enhanced BK-β1 expression and BK-β1-mediated channel activation by MG132. A, protein expression of BK-β1 in STZ-induced diabetic aortas with or without 24-h treatment of MG132 (10 μm). Inhibition of proteasomal activity by MG132 increased BK-β1 protein expression in diabetic vessels. B, robust BK channel openings by DHS-1 (0.1 μm) were observed in freshly isolated diabetic coronary SMCs after 12-h incubation with MG132. Group data with statistical analysis are shown in the bar graphs. Dashed lines represent the channel closed state (c). W/O, washout.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of MuRF1 and BK-β1 expression by NF-κB. A, protein expression of NF-κB1/p105, NF-κB1/p50, and RelA/p-65 in the aortas of control and STZ-induced diabetic mice. The levels of NF-κB/p105, NF-κB1/p50, and p-65 protein expression were significantly increased in diabetic mice compared with controls (Ctrl). B, 24-h incubation with 0.5 μm TPCA-1 suppressed the levels of NF-κB1/p105, NF-κB1/p50, and MuRF1 expression but markedly increased that of IκB-β in both NG- and HG-cultured human coronary SMCs. TPCA-1 treatment did not alter BK-β1 expression in NG-cultured cells but significantly enhanced that in HG-cultured cells. Group data and statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Control, Incubation, Cell Culture

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Flow cytometry gates for cell viability were set based on living and EtOH-killed controls (A). Incubation for 2 h (grey bar) with pDNA had no impact on vascular smooth muscle cell (vSMC) viability. Fifty-percent pCsM (C) and Cs (D) stabilize vSMC viability by 12 h post-incubation, whereas Vehicle (E) exposure drives protracted declines. Cell viability was unchanged 24 h after 20% treatments (F). (*p≤0.01, §p≤0.05)

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry, Incubation

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Flow cytometry gates to determine Live CD59-positive vSMC were set based on living and EtOH-killed controls (A). Chitosan (pCsM, CsM, Cs) initially decreased the number of CD59-positive cells (B, p≤0.001), an effect that was rapidly, potently and durably reversed, especially in the case of Cs (C, *p≤0.01, §p≤0.05).

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry

Journal: Carbohydrate polymers

Article Title: Stability and Bioactivity of Chitosan as a Transfection Agent in Primary Human Cell Cultures: A Case for Chitosan-only Controls

doi: 10.1016/j.carbpol.2017.10.021

Figure Lengend Snippet: Mean fluorescent intensity from flow cytometry was used to approximate average CD59 expression vSMC (A). CD59 is transiently depressed by pCsM, CsM and Cs (B, p≤0.001) but recovers to baseline by 24 h despite the presence of Vehicle (C). In the absence of chitosan, Vehicle decreases CD59 expression.

Article Snippet: 2.2.1 Culture Primary human vSMC from ScienCell (Catalog #1100, Carlsbad, California) were cultured under standard conditions (5% CO 2 at 37 °C) in Medium 231 containing 5% Smooth Muscle Growth Supplement (Gibco), 0.1 mg/mL Primocin (Invivogen, San Diego, California), and 100 U/mL each penicillin and streptomycin (Life Technologies, Thermo Fisher Scientific) in BioCoat Collagen I culture flasks (Corning, New York).

Techniques: Flow Cytometry, Expressing