Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells

doi: 10.1161/atvbaha.111.235721

Figure Lengend Snippet: Figure 1. Circulating CCL20 levels in normo- and hypercholes- terolemic subjects. Circulating CCL20 levels in normocholester- olemic (total cholesterol 239 mg/dL; n54) and hypercholes- terolemic subjects (total cholesterol 240 mg/dL; n53). Data are expressed as meanSEM (P0.01: *vs normocholesterol- emic subjects).

Article Snippet: CCL20 recombinant protein (40 pg/mL) (Boster Biological Technology, LTD) was used as a positive control and cell supernatant from non-stimulated cells as a control of spontaneous migration.

Techniques:

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells

doi: 10.1161/atvbaha.111.235721

Figure Lengend Snippet: Figure 2. LDL induce the secretion of biologically relevant levels of CCL20 protein in VSMC. A, Vascular smooth muscle cells (VSMC) were stimulated with LDL (300 g/mL for 24 hours) and CCL20 release was assessed by EIA (n6; P0.05: *vs controls). B, Quantita- tive data from cell migration assays of human lymphocytes induced by supernatants from human VSMC cultured under control condi- tions (control), supernatants from human VSMC treated with LDL (csLDL; 300 g/mL for 24 hours) or human recombinant CCL20 (hrCCL20; 40 pg/mL in the bottom chamber [B]) (n6; P0.05: *vs controls). As a negative control CCL20 was added only in the top chamber (T) or in both chambers (T/B) of the transwell system. C, Representative images showing lymphocyte migration. D, VSMC were transfected with a CCL20 specific siRNA (siCCL20) or a control siRNA (siRAND), and the effect of LDL treatment on CCL20 mRNA levels, CCL20 secretion, and lymphocyte migration induced by cell supernatants were determined [n6; P0.05: *vs controls (unstimulated cells transfected with siRAND); #versus cells transfected with siRAND and stimulated with LDL].

Article Snippet: CCL20 recombinant protein (40 pg/mL) (Boster Biological Technology, LTD) was used as a positive control and cell supernatant from non-stimulated cells as a control of spontaneous migration.

Techniques: Migration, Cell Culture, Control, Recombinant, Negative Control, Transfection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells

doi: 10.1161/atvbaha.111.235721

Figure Lengend Snippet: Figure 3. LDL induce CCL20 expression through a transcriptional mechanism that involves NF-B. A, Vascular smooth muscle cells (VSMC) were induced with 300 g/mL LDL for 4 hours in the presence or in the absence of 5,6-dicloro-1-(b-D-ribofuranosil)- benzimidazol (DRB) and CCL20 expression was analyzed (n9; P0.05: *vs control cells; #vs cells treated with LDL alone). B, VSMC were transfected with the pCCL20/-2007 construct and treated with LDL (300 g/mL for 7 hours) (n9; P0.05: *vs control cells). C, Effect of NDGA, parthenolide (Part) and BAY 11-7082 (BAY) on CCL20 mRNA levels induced by LDL (300 g/mL for 4 hours) (n9; P0.05: *vs controls; #vs LDL alone). D, VSMC were treated with LDL (300 g/mL) in the presence or absence of parthenolide, and cytosolic or nuclear extracts were analyzed by Western blot. Representative immunoblots using antibodies against IB and p65 are shown. Beta-actin and nucleolin (Nucl.) were used as a loading control for citosolic and nuclear extracts, respectively. E, Confocal microscopy analysis showing the mobilization of p65 to the nuclei in cells stimulated with LDL and the preventive effect exerted by par- thenolide (LDL/Part).

Article Snippet: CCL20 recombinant protein (40 pg/mL) (Boster Biological Technology, LTD) was used as a positive control and cell supernatant from non-stimulated cells as a control of spontaneous migration.

Techniques: Expressing, Control, Transfection, Construct, Western Blot, Confocal Microscopy

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells

doi: 10.1161/atvbaha.111.235721

Figure Lengend Snippet: Figure 4. NF-B is involved in LDL-induced CCL20 upregulation. A, Vascular smooth muscle cells (VSMC) were transiently transfected with various CCL20 promoter deletion mutants and promoter activity in the absence (white bars) or presence of LDL (black bars; 300 g/mL for 7 hours) was assessed. The location of the putative response elements is indicated. The activity of pCCL20/-117 mutated in the NF-B site (80/71; deleted white circle) is also shown (n7; P0.05: *vs control cells transfected with the same construct). B, Schematic representation of pCCL20/-165. The core consensus of the NF-B site is indicated in bold, and changes introduced by mutagenesis are boxed. C, Representative autoradiogram of EMSA performed with the CCL20-88/-65 probe and nuclear protein extracts from controls and cells treated with LDL in the presence or absence of 1 mol/L parthenolide (Part). The position of 4 com- plexes (I to IV), whose upregulation by LDL was prevented by parthenolide (left panel) and competed by a molar excess of unlabeled probe (100-fold) (middle panel) is indicated. The supershifted bands on addition of a specific antibody against p65 (antip65) are indi- cated (right panel; double arrowhead). D, Chromatin immunoprecipitation (ChIP) from control cells and cells treated with LDL (300 g/mL for 2 hours) using an antip65 antibody (IP: p65) or a nonspecific rabbit IgG (IP: IgG). Top: The enrichment of NFB was quanti- fied by real-time PCR using CCL20 promoter specific primers. Data were normalized to the total input DNA and are represented as meansSEM of 2 independent experiments performed in duplicate (P0.05: *vs controls). Bottom: Agarose gel electrophoresis of PCR products.

Article Snippet: CCL20 recombinant protein (40 pg/mL) (Boster Biological Technology, LTD) was used as a positive control and cell supernatant from non-stimulated cells as a control of spontaneous migration.

Techniques: Transfection, Activity Assay, Control, Construct, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL20 Is Increased in Hypercholesterolemic Subjects and Is Upregulated By LDL in Vascular Smooth Muscle Cells

doi: 10.1161/atvbaha.111.235721

Figure Lengend Snippet: Figure 5. Atherosclerotic coronary arteries show increased CCL20 mRNA levels and NF-B activity. A, mRNA levels of CCL20, CCR6, and monocyte chemotactic protein 1 (MCP-1) in human nonatherosclerotic coronary arteries (non-ATC; n7; white bars) and human atherosclerotic coronary arteries (ATC; n7; black bars). Data are expressed as meanSEM (P0.05: *vs non-ATC). B, Representative EMSA performed with the CCL20-88/-65 probe and whole-protein extracts from non-ATC (n6) and ATC (n6). Arrowheads indicate the main complexes upregulated in ATC and competed with a molar excess of unlabeled probe (100). C, High-power views showing immunostaining corresponding to CCL20, -SMA (marker of VSMC), and the merge image (nuclei staining in blue) demonstrating their colocalization in the intima of a representative ATC (n4). CT indicates control immunostaining of a consecutive section incubated with nonimmune goat IgG. Bar20 m. D, CCL20 levels in conditioned media from human vascular tissues. Intima-media layers from carotid endarterectomy specimens (atheroma; n15) and healthy aortas (control; n8) were incubated in serum-free medium (24 hours at 37°C), and CCL20 levels were analyzed by EIA. CCL20 levels were normalized by protein concentration. Data are expressed as meanSEM (*P0.001 vs aorta controls).

Article Snippet: CCL20 recombinant protein (40 pg/mL) (Boster Biological Technology, LTD) was used as a positive control and cell supernatant from non-stimulated cells as a control of spontaneous migration.

Techniques: Activity Assay, Immunostaining, Marker, Staining, Control, Incubation, Protein Concentration

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2024.103481

Figure Lengend Snippet:

Article Snippet: Human hematopoietic stem cell expansion cytokine package (IL-6) (100 μg SCF, 100 μg FLT3 ligand, 100 μg TPO, 20 mg IL-6) , PeproTech , Cat# HHSC6.

Techniques: Cell Culture, Recombinant, Red Blood Cell Lysis, Reverse Transcription, TA Cloning, Software

Journal: STAR Protocols

Article Title: Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells

doi: 10.1016/j.xpro.2024.103481

Figure Lengend Snippet: Medium for UCB HSPCs

Article Snippet: Human hematopoietic stem cell expansion cytokine package (IL-6) (100 μg SCF, 100 μg FLT3 ligand, 100 μg TPO, 20 mg IL-6) , PeproTech , Cat# HHSC6.

Techniques: Concentration Assay