Journal: Journal of Bacteriology

Article Title: Identification and Characterization of a New Ferric Enterobactin Receptor, CfrB, in Campylobacter ▿ †

doi: 10.1128/JB.00478-10

Figure Lengend Snippet: Critical role of CfrB in Campylobacter-host interaction. (A) Effect of the CfrB mutation on colonization of chickens by C. jejuni JL11 and C. coli JL170. The experiment included two groups, and chickens in each group (15 birds) were inoculated with a 1:1 mixture of a specific Campylobacter strain and its isogenic cfrB mutant. Fecal samples were collected by cloacal swabs and used for CFU enumeration. Each bar represents the mean log transformed CFU of each strain in each group at the indicated days postinoculation (DPI). The broken line indicates the limit of detection. (B and C) Inactivation of CfrB abolishes C. coli JL170 colonization of chickens. The experiment included three groups of birds. The first two groups of birds were inoculated with JL170 or its isogenic cfrB mutant (JL170 CfrB−) (B), while the third group was infected with a 1:1 mixture of the two strains (C). For each group, six birds were euthanized and the cecal contents were collected at the indicated DPI. Each symbol indicates the log number of CFU/g of cecal contents for a single chicken. The horizontal bars indicate the means of groups at the indicated times. The broken line indicates the limit of detection. (D) Inhibitory effect of CfrB-specific antiserum on FeEnt-mediated growth promotion in Campylobacter. CfrB antiserum, MOMP antiserum, preimmune serum, or PBS was mixed with JL11 and JL170 cells for a modified growth promotion assay as described in Materials and Methods.

Article Snippet: Prior to the assay, the CfrB-specific rabbit antiserum, the preimmune control rabbit serum, and the major outer membrane protein (MOMP) rabbit antiserum specific for Campylobacter (kindly provided by Qijing Zhang, Iowa State University) were heat inactivated at 56°C for 30 min. To evaluate the inhibitory effect of anti-CfrB serum on CfrB function, 1 ml of MH broth grown log-phase culture of each Campylobacter strain was mixed with 1 ml of either CfrB antiserum, preimmune serum, MOMP antiserum, or phosphate-buffered saline (PBS) and then added to 23 ml of MH agar containing 20 μM DFO and poured into petri dishes for solidification.

Techniques: Mutagenesis, Transformation Assay, Infection, Modification

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: Mapping of the USF responsive element. Left, full-length PHA and its mutated or deleted constructs are represented. The numbers indicate the position of the deleted fragments relative to the start of translation. Right, the different constructs were transiently transfected into keratinocytes and NIH-3T3 cells together with or without a USF-1 expression vector. Luciferase activities were assayed as described in the Materials and Methods and are shown as absolute values. Error bars reflect the SD between at least three separate experiments performed in triplicate.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Construct, Transfection, Expressing, Plasmid Preparation, Luciferase

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: Identity of nuclear protein bound to the lama3 USF-responsive element. The 21 bp sequence containing the heptameric E-box (wt) is incubated with nuclear extracts from NIH-3T3 cells (A) and keratinocytes (B) transfected with HA-tagged USF-1. When indicated, nuclear extracts are incubated with a pre-immune (pi) serum or with antibodies raised against USF-1 or HA. Ebox3 probe in which the heptameric E-box was replaced by a BamHI site (mt) or the canonical E-box probe (Ebox) was incubated with USF-1-expressing NIH-3T3 nuclear extracts as indicated in lanes 9 and 10, respectively.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Sequencing, Incubation, Transfection, Expressing

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: Among the bHLHzip members tested, only USF releases the cell-specific inhibition of lama3 gene expression. NIH 3T3 fibroblasts were transfected with the 6.2H or 3USB-tk constructs along with either USF-1, Max, Myc or Max/Myc and assayed as described in Materials and Methods. The values (fold stimulation) are derived from the ratio between the basal and the induced promoter activities. Error bars reflect the SD between three separate experiments performed in triplicate.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Inhibition, Expressing, Transfection, Construct, Derivative Assay

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: UV irradiation increases binding activity of USF-1 on the lama3 non-canonical E-box and induced 6.2H construct promoter activity. (A) USF binding analysis after UV irradiation. NIH-3T3 were UV irradiated (40 J/m2) and nuclear extracts were prepared at 30 min, 1, 2 and 3 h post-irradiation. Unstimulated and UV-irradiated nuclear extracts were then incubated with the radiolabeled Ebox3 probe. (B) 6.2H and empty vector control constructs were transiently transfected into NIH-3T3 fibroblasts. Twenty-four hours after transfection, the cells were UV irradiated (40 J/m2) and harvested at 30 min, 1, 2 and 3 h post-irradiation for luciferase activity measurements.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Irradiation, Binding Assay, Activity Assay, Construct, Incubation, Plasmid Preparation, Transfection, Luciferase

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: UV and USF inducibility of the lama3 promoter in fibroblasts. (A) 6.2H and mutEbox3 constructs were transiently transfected into NIH-3T3 fibroblasts and keratinocytes stimulated by UV irradiation or transfected with a USF-1 expression vector. Luciferase activities were determined as described in Materials and Methods. Empty vector luciferase values correspond to the background expression of the pGL2 (no promoter) reporter gene. The luciferase activity values and error bars reflect the average and the SD between at least three separate experiments performed in triplicate. (B) USF-1 binds the lama3 promoter in vivo. Untreated NIH-3T3 cells (lanes 1 and 3) or UV-irradiated NIH-3T3 cells (lanes 2 and 4) were chemically cross-linked and immunoprecipitated with a specific USF-1 antibody or a preimmune control (pi) serum. Lane ct corresponds to the PCR control directly amplified from PHA. (C) Analysis of lama3 mRNA expression level by semi-quantitative RT–PCR assay using total RNA extracted from non-stimulated (lane 4), UV-stimulated (lane 3) and USF-1-transfected (lane 2) NIH-3T3 cells (3T3) as templates. The Lama3 mRNA expression level in keratinocytes (K) is shown as a positive control (lane 1). Bottom, amplification of GAPDH as an internal control.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Construct, Transfection, Irradiation, Expressing, Plasmid Preparation, Luciferase, Activity Assay, In Vivo, Immunoprecipitation, Amplification, Quantitative RT-PCR, Positive Control

Journal:

Article Title: Binding of USF to a non-canonical E-box following stress results in a cell-specific derepression of the lama3 gene

doi:

Figure Lengend Snippet: Functional analysis of the AP-1 binding sites and the intervening sequences within PHA. Full-length, truncated and AP-1-mutated PHA constructs are represented on the left. Keratinocytes and NIH-3T3 cells were transfected with pRK7 USF-1 along with different truncated/mutated constructs derived from PHA. Mut ABC construct corresponds to pHA in which the AP-1 binding sites have been mutated; Δ60 and Δ109 constructs correspond, respectively, to a 60 and 109 bp deletion between the AP1-A and AP1-B binding sites. 109 mix construct represents Δ60 in which 49 bp were inserted in a random order to respect the 109 bp spacing between AP-1 sites. The pCDNA3 construct corresponds to the substitution of 109 nt residing between AP1-A and AP1-B with a 109 bp heterologous sequence from the pCDNA3 polylinker plasmid.

Article Snippet: The chromatin was sheared by sonication and USF-1-containing complexes were recovered by immunoprecipitation using USF-1-specific serum (USF-1 antibody sc-229; Santa Cruz) or preimmune serum as a control.

Techniques: Functional Assay, Binding Assay, Construct, Transfection, Derivative Assay, Sequencing, Plasmid Preparation

Journal: Genes & Cancer

Article Title: Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

doi: 10.1177/1947601911432495

Figure Lengend Snippet: Oligonucleotides Used for siRNA and PCR Experiments

Article Snippet: After blocking with 10% preimmune goat serum in PBS for 45 minutes, slides were incubated with p21-specific monoclonal antibody (1:200) (Pharmingen, San Diego, CA) for 1 hour at room temperature.

Techniques:

Journal: Genes & Cancer

Article Title: Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

doi: 10.1177/1947601911432495

Figure Lengend Snippet: Silencing of p21 prevents FDH-induced G1 arrest and accelerates apoptosis in A549 cells. (A) Levels of p21 mRNA in A549 cells after transfection with p21-specific siRNA and FDH induction. (B) Levels of FDH and p21 proteins (Western blot) at different times of FDH induction in untreated (control), treated with scrambled siRNA (Scr), and treated with p21-specific siRNA (siRNA). Actin is shown as loading control. (C) Distribution of cells between cell cycle phases in p21-proficient cells without FDH (top), p21-proficient cells after FDH induction (middle), and p21-silenced cells after FDH induction (bottom). Assay was carried out at 48 hours after FDH induction. (D) Apoptosis evaluated by annexin V/PI staining in p21-proficient (scrambled) and silenced (siRNA) cells in the presence (+) and absence (–) of FDH. (E) Calculation of apoptotic cells (% of the total cells) from D. Error bars represent ±SD (n = 3).

Article Snippet: After blocking with 10% preimmune goat serum in PBS for 45 minutes, slides were incubated with p21-specific monoclonal antibody (1:200) (Pharmingen, San Diego, CA) for 1 hour at room temperature.

Techniques: Transfection, Western Blot, Staining

Journal: Genes & Cancer

Article Title: Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

doi: 10.1177/1947601911432495

Figure Lengend Snippet: FDH-induced antiproliferative effects are different in HCT116 p21+/+ and p21−/− cells. (A) Levels of FDH and p21 proteins (Western blot) at different times after FDH transfection. Actin is shown as loading control. (B) Apoptosis evaluated by annexin V/PI staining in p21+/+ and p21−/− cells in the presence (+) and absence (–) of FDH. Time after FDH transfection is indicated. (C) Calculation of apoptotic cells (% of the total cells) from B. (D) Distribution of HCT116 p21+/+ and p21−/− cells between cell cycle phases at 48 hours after FDH transfection. Error bars represent ±SD. For statistical analysis, a Student t test was performed (*P < 0.05, n = 3).

Article Snippet: After blocking with 10% preimmune goat serum in PBS for 45 minutes, slides were incubated with p21-specific monoclonal antibody (1:200) (Pharmingen, San Diego, CA) for 1 hour at room temperature.

Techniques: Western Blot, Transfection, Staining

Journal: Genes & Cancer

Article Title: Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

doi: 10.1177/1947601911432495

Figure Lengend Snippet: Subcellular distribution of p21 in FDH-deficient and FDH-expressing A549 and HCT116 cells assessed by confocal microscopy. (A) Green: p21 protein stained with specific antibody and secondary antibody conjugated with Alexa Fluor 488; red: nuclei stained with TO-PRO-3 iodide dye; yellow: colocalization. (B) Green: p21 protein stained with specific antibody and secondary antibody conjugated with Alexa Fluor 488; red: fibrillarin stained with specific antibody and secondary antibody conjugated with Alexa Fluor 555; yellow: colocalization.

Article Snippet: After blocking with 10% preimmune goat serum in PBS for 45 minutes, slides were incubated with p21-specific monoclonal antibody (1:200) (Pharmingen, San Diego, CA) for 1 hour at room temperature.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Genes & Cancer

Article Title: Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress

doi: 10.1177/1947601911432495

Figure Lengend Snippet: Schematic depicting the FDH anti-proliferative cascade. FDH depletes the 10-fTHF pool, leading to inhibition of the de novo purine biosynthesis and depletion of purines in the cell. This activates the p53 protein, which in turn induces transcriptional activation of p21 and proapoptotic PUMA. Accumulated p21 protein activates either G1 or G2 arrest, both of which delay apoptosis.

Article Snippet: After blocking with 10% preimmune goat serum in PBS for 45 minutes, slides were incubated with p21-specific monoclonal antibody (1:200) (Pharmingen, San Diego, CA) for 1 hour at room temperature.

Techniques: Inhibition, Activation Assay

Journal: Vaccines

Article Title: Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas

doi: 10.3390/vaccines10071074

Figure Lengend Snippet: Anti-pentamer rabbit immune sera neutralize HCMV infection of primary placental cell types. Sera from rabbits immunized with RBT pentamer described in were tested for ability to neutralize infection of placental cell types by the clinical HCMV strain VR1814. For comparison, hyperimmune globulin (Cytogam) and non-immune sera were also evaluated. Fifty percent neutralization titers (NT50s) were calculated for each serum sample and plotted for each of the following cell types: ( A ) Human placental fibroblasts (HPFs) from an 8-week-gestation placenta, ( B ) trophoblast progenitor cells (TBPCs) from a 15.6-week-gestation placenta, ( C ) amniotic epithelial cells (AmEpCs) from a 21-week-gestation placenta, ( D ) primary human cytotrophoblasts (CTBs) from a 16-week-gestation placenta, ( E ) CTBs from a 20.1-week-gestation placenta, and ( F ) CTBs from a 20.2-week-gestation placenta. GA, gestational age. #, NT50 < 10. ##, NT50 > 10 5 , NT90 = 4.1 × 10 4 . ###, NT50 > 10 5 , NT90 > 10 5 .

Article Snippet: The stock virus was preincubated with media alone (no serum control), heat-inactivated preimmune serum (negative serum), or Cytogam, an HCMV hyperimmune globulin preparation (CSL Behring LLC, King of Prussia, PA, USA), at the indicated final dilutions for 1 h at 37 °C with moderate agitation.

Techniques: Infection, Comparison, Neutralization