Journal: Infection and Immunity

Article Title: Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

doi: 10.1128/iai.68.7.4282-4288.2000

Figure Lengend Snippet: FIG. 1. Expression of MCP-1 mRNA in HUVEC infected with C. pneu- moniae or C. trachomatis. (A) RNA was isolated at 0, 1, 2, 4, 8, 12, and 24 h of incubation, and levels of MCP-1 mRNA were determined by RT-PCR as de- scribed in Materials and Methods. (Top panel) HUVEC were incubated with medium alone (uninfected) or crude lysates of HEp-2 cells (mock infected). (Middle panel) HUVEC were infected with C. pneumoniae A-03 at an MOI of 1:1 or treated with 500 U of TNF-a per ml as a positive control. (Bottom panel) Infection with C. trachomatis L2. The inoculum was equivalent to that of C. pneumoniae A-03. M, molecular size markers. (B) Levels of MCP-1 mRNA following infection of HUVEC with C. pneumoniae (Cp) or C. trachomatis (Ctr) were measured by plotting the densitometric MCP-1/b-actin RT-PCR product ratios. Data points represent the means 6 standard errors of the mean of five separate experiments. p, P , 0.01.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC 1730-CRL) were cultured in 75-cm2 flasks and maintained in Ham’s F12K medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-amphotericin B (Fungizone) mix, 30 mg of endothelial cell growth supplement per ml, and 100 mg of heparin (Sigma, St. Louis, Mo.) per ml.

Techniques: Expressing, Infection, Isolation, Incubation, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Infection and Immunity

Article Title: Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

doi: 10.1128/iai.68.7.4282-4288.2000

Figure Lengend Snippet: FIG. 2. Translocation of NF-kB determined by EMSA in HUVEC infected with C. pneumoniae or C. trachomatis. Mock-infected HUVEC were incubated with crude lysates of HEp-2 cells. Infection with C. pneumoniae A-03 or C. trachomatis L2 was performed at an MOI of 1:1. Treatment of HUVEC with 500 U of TNF-a per ml served as a positive control. Nuclear extracts were prepared after 30 min of incubation. EMSAs were performed with a 32P-labeled oligonu- cleotide containing the NF-kB consensus sequence (see Materials and Methods). C. pneumoniae A-03 and TNF-a caused an increase in NF-kB DNA-binding activities of nuclear protein complexes compared to the levels in mock-infected cells and C. trachomatis L2-infected cells.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC 1730-CRL) were cultured in 75-cm2 flasks and maintained in Ham’s F12K medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-amphotericin B (Fungizone) mix, 30 mg of endothelial cell growth supplement per ml, and 100 mg of heparin (Sigma, St. Louis, Mo.) per ml.

Techniques: Translocation Assay, Infection, Incubation, Positive Control, Labeling, Sequencing, Binding Assay

Journal: Infection and Immunity

Article Title: Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

doi: 10.1128/iai.68.7.4282-4288.2000

Figure Lengend Snippet: FIG. 3. Supershift analysis of NF-kB-binding complexes. DNA-binding reac- tions with nuclear extracts from HUVEC infected with C. pneumoniae A-03 were incubated with 32P-labeled NF-kB oligonucleotide in the presence of antibodies (Ab) to NF-kB proteins p50 (NF-kB1), p52 (NF-kB2), p65 (RelA), p68 (RelB), and p75 (c-Rel). The solid arrowhead indicates the NF-kB band. Supershifts of p50 and p65 are indicated by the open and solid arrows, respectively.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC 1730-CRL) were cultured in 75-cm2 flasks and maintained in Ham’s F12K medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-amphotericin B (Fungizone) mix, 30 mg of endothelial cell growth supplement per ml, and 100 mg of heparin (Sigma, St. Louis, Mo.) per ml.

Techniques: Binding Assay, Infection, Incubation, Labeling

Journal: Infection and Immunity

Article Title: Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

doi: 10.1128/iai.68.7.4282-4288.2000

Figure Lengend Snippet: FIG. 4. Immunofluorescent staining of NF-kB p65 in HUVEC infected with C. pneumoniae or C. trachomatis. HUVEC grown on gelatin-coated glass coverslips were infected with C. pneumoniae A-03 or C. trachomatis L2 at an MOI of 1:1. After 30 min of infection, cells were processed for immunofluorescent staining as described in Materials and Methods with rabbit polyclonal antibody against the p65 component of NF-kB. Stained cells were immediately examined by confocal microscopy (31,000). Absence of nuclear NF-kB p65 is observed in uninfected, mock-infected, and C. trachomatis L2-infected cells (panels A, B, and D, respectively). Nuclear localization of NF-kB is observed in HUVEC infected with C. pneumoniae A-03 (panel C) and to a greater extent in cells treated with 500 U of TNF-a per ml for 30 min as a positive control (panel E). Panel F represents a control to rule out possible nonspecific binding of the FITC-conjugated anti-rabbit IgG in C. pneumoniae A-03-infected HUVEC.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC 1730-CRL) were cultured in 75-cm2 flasks and maintained in Ham’s F12K medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-amphotericin B (Fungizone) mix, 30 mg of endothelial cell growth supplement per ml, and 100 mg of heparin (Sigma, St. Louis, Mo.) per ml.

Techniques: Staining, Infection, Confocal Microscopy, Positive Control, Control, Binding Assay

Journal: Infection and Immunity

Article Title: Requirement for NF-κB in Transcriptional Activation of Monocyte Chemotactic Protein 1 by Chlamydia pneumoniae in Human Endothelial Cells

doi: 10.1128/iai.68.7.4282-4288.2000

Figure Lengend Snippet: FIG. 5. Effects of parthenolide on NF-kB activation and MCP-1 gene ex- pression in HUVEC infected with C. pneumoniae. (Top panel) Prior to infection, HUVEC grown on six-well plates were preincubated with 50 mM parthenolide for 1 h. Cells were then infected with C. pneumoniae A-03 at an MOI of 1:1 or treated with 500 U of TNF-a per ml in medium containing 0 or 50 mM parthe- nolide for 30 min before nuclear protein extraction. NF-kB activation was ex- amined by EMSA. (Bottom panel) HUVEC grown on 24-well plates were pre- incubated with 50 mM parthenolide for 1 h. Cells were then infected with C. pneumoniae A-03 at an MOI of 1:1 or treated with 500 U of TNF-a per ml in medium containing 0 or 50 mM parthenolide for 1 h before total RNA isolation. Expression of MCP-1 mRNA was examined by RT-PCR.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC 1730-CRL) were cultured in 75-cm2 flasks and maintained in Ham’s F12K medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-amphotericin B (Fungizone) mix, 30 mg of endothelial cell growth supplement per ml, and 100 mg of heparin (Sigma, St. Louis, Mo.) per ml.

Techniques: Activation Assay, Infection, Protein Extraction, Incubation, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction