Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) VSMCs were grown in serum for 48 hours and then harvested for RNA. Coro1B,1C, and GAPDH mRNA was detected using sequence specific PCR primers. Rat lung cDNA was used as a positive control. (B) VSMCs were transfected with siNegCtrl, siCoro1B or siCoro1C with the Amaxa electroporation system. Cell lysates were harvested and immunoblotted for Coro1B and Coro1C and CDK4 as a loading control. On average Coro1B was down regulated by 87 ± 3% (N= 10) and Coro1C by 68 ± 5% (N=5). (C) Live cell imaging was used to measure the distance traveled by VSMCs transfected with siNegCtrl, siCoro1B, or Coro 1C, cells were serum starved for 24hrs and then stimulated with 10ng/ml of PDGF for 12hrs. The graph represents the mean ± SEM of the distance traveled by each cell. The N (number of cells) is indicated within each column. (*p< 0.001, PDGF treated siNegCtrl compared to siCoro1B).

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Sequencing, Positive Control, Transfection, Electroporation, Live Cell Imaging

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) Confocal images were acquired after transfecting VSMCs with siNegCtrl or siCoro1B using the Amaxa electroporation system. Cells were serum starved for 48 hrs and then fixed and permeabilized. Immunofluorescence of Coro1B (red), DAPI (blue) and phalloidin (green) was then detected using the Zeiss LSM 510 META Laser Scanning Confocal Microscope System. (B) VSMCs were transfected with empty vector or Myc- Coro1B with the Amaxa electroporation system, serum starved for 48 hrs and then fixed and permeabilized. Immunofluorescent images using DAPI (blue), phalloidin (green), Coro1B (red) and Myc (magenta) antibodies were then acquired.

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Electroporation, Immunofluorescence, Microscopy, Transfection, Plasmid Preparation

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) VSMCs were serum starved for 48 hours, and then stimulated with 10 ng/ ml PDGF for the indicated times. Cell lysates were harvested and immunoblotted with Coro1B and p-Ser2 Coro1B antibodies. The graph represents the mean ± SEM of densiometric analysis from 3–5 independent experiments. (*p< 0.01, compared to control). (B) VSMCs were serum starved for 48 hrs, stimulated with 10ng/ml PDGF for 15 minutes and then fixed and permeabilized. Immunofluorescent images using DAPI (blue), phalloidin (green) and p-Ser2 Coro1B antibody (red) were then acquired acquired using the Zeiss LSM 510 META Laser Scanning Confocal Microscope System.

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Microscopy

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) VSMCs were serum starved for 48 hours, and then pre-incubated with 0.5 μM Ro-32-0432 (PKC inhibitor) or vehicle for 30 min. The cells were then stimulated with 10 ng/ml PDGF for 5 minutes. Cell lysates were harvested and immunoblotted with Coro1B and p-Ser2 Coro1B antibody. The graph represents the mean ± SEM of densiometric analysis from 4 independent experiments. (*p< 0.01 compared to control, = p<0.0001 compared to control PDGF treated). (B) VSMCs were transfected with siNegCtrl or siPKCε using the Amaxa electroporation system. Cells were serum starved for 48 hours and then stimulated with 10 ng/ml PDGF for 5 minutes. Cell protein lysates were harvested and immunoblotted with p-Ser2 Coro1B, Coro1B and PKCε antibodies. Blots presented are from the same gel, but unrelated lanes were removed for clarity. The graph represents the mean ± SEM of densiometric analysis from 4 independent experiments. (*p< 0.0001, siNeg Ctrl vs. PDGF stimulated siNegCtrl, and = p< 0.01, PDGF stimulated siNegCtrl vs, PDGF stimulated siPKCε transfected).

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Incubation, Transfection, Electroporation

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: VSMCs were transfected with the Myc tagged Coro1B WT or S2A phospho-deficient mutant using the Amaxa electroporation system and then serum starved for 24hrs. Cells were harvested and migration was assessed via modified Boyden chamber assay (A). This graph represents the mean ± SEM of the number of cells migrated per field (*p< 0.001, pcDNA3 vs pcDNA3 with PDGF stimulation, = p < 0.01, PDGF stimulated pcDNA3 vs. PDGF stimulated Coro1B WT transfected cells, and # p < 0.001, PDGF stimulated pcDNA3 vs. PDGF stimulated Coro1B S2A mutant transfected cells N=4). (B) Protein lysates from excess cells not used in the migration assay were harvested and immunoblotted with Myc and Coro1B to verify Coro1B WT and S2A protein expression. (C) VSMCs were transfected with Myc tagged S2A Coro1B, serum starved for 24 hrs, then fixed and permeabilized. Immunofluorescent images using DAPI (blue), phalloidin (green), Coro1B antibody (red) and Myc antibody (magenta) were then acquired acquired using the Zeiss LSM 510 META Laser Scanning Confocal Microscope System.

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Transfection, Mutagenesis, Electroporation, Migration, Modification, Boyden Chamber Assay, Expressing, Microscopy

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) VSMCs were serum starved for 24 hrs and then treated with 10 ng/ml PDGF for the indicated times. Cell lysates were harvested and immunoprecipitated with Coro1B antibody. Membranes were immunoblotted with Coro1B and ARPC2. The graph represents the mean ± SEM of densiometric analysis from 3–4 independent experiments. Data are plotted as total ARPC2/ Coro1B. (*p< 0.05 compared to control). (B) VSMCs were transfected with the Myc tagged Coro1B WT, the S2A Coro1B phospho-deficient mutant, or the S2D Coro1B phospho-mimetic mutant using the Amaxa electroporation system and then serum starved for 24hrs. Cell lysates were harvested and immunoprecipitated with Myc antibody. Membranes were immunoblotted with anti-Myc and ARPC2. The graph represents the mean ± SEM of densiometric analysis from 4 independent experiments. Data are plotted as total ARPC2/Myc. (+ p< 0.05 compared to S2A).

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Immunoprecipitation, Transfection, Mutagenesis, Electroporation

Journal: Circulation research

Article Title: Role of Coronin 1B in PDGF-induced Migration of Vascular Smooth Muscle Cells

doi: 10.1161/CIRCRESAHA.111.255745

Figure Lengend Snippet: (A) Mice were subjected to left carotid wire injury and then sacrificed 7 or 14 days post surgery. The left carotids were collected and the right carotid was used as a control. Tissue was harvested for protein and immunoblotted with p-Ser2 Coro1B and Coro1B antibodies (N=3 *p< 0.05 compared to control). (B) Sprague-Dawley rats were subjected to balloon injury of the left common carotid artery. Ten days after injury, arteries were harvested, embedded in OTC and then cut into 7μm sections. Sections were stained for p-Ser2 Coro1B (magenta, panels a and b) or α smooth muscle actin (red, panels c and d) and nuclei (DAPI, blue, all panels) Green represents the autofluorescence of the internal elastic lamina. The blood vessel is labeled as follows; I denotes intimal region, M denotes medial region, and A denotes the adventitia.

Article Snippet: Plasmid Construction and Site- directed Mutagenesis C-terminal Myc-tagged Coro1B-WT pCDNA3 was constructed by amplifying the open reading frame of human Coro1B from pCMV6-AC-Coro1B (Origene) using Phusion High-Fidelity DNA polymerase (Thermo-Fisher Scientific) as well as primers that contained the Myc coding sequence.

Techniques: Staining, Labeling