Journal: PLoS Pathogens

Article Title: Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

doi: 10.1371/journal.ppat.1003145

Figure Lengend Snippet: (A) Transcriptional activation of CEC1 promoter upon PGN challenge. The graph reports the z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments. (B and C) Transcriptional regulation of LRIM1 promoter (B) upon PGN challenge (z-scores calculated from the ratios of averaged RLU measurements after PGN and PBS treatments) and (C) in basal conditions (z-scores calculated from the RLU measured after PBS treatments). Positive hits are shown as open circles.

Article Snippet: Four days after dsRNA treatment, cells were washed and co-transfected in 50 μl of final volume with CEC1 or LRIM1 promoter firefly luciferace fusion reporter constructs (Promega pGL3 backbone) and reference pAct5C-Renilla luciferase construct using Effectene Transfection Reagent (QIAGEN) following the manufacturer's instructions.

Techniques: Activation Assay

Journal: PLoS Pathogens

Article Title: Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

doi: 10.1371/journal.ppat.1003145

Figure Lengend Snippet: A total of 22 positive (green) and negative (red) regulators are shown. Dotted lines indicate modulations of CEC1 and LRIM1 gene expression upon PGN challenge, and solid lines describe the effect of genes on phagocytosis and basal expression of LRIM1 .

Article Snippet: Four days after dsRNA treatment, cells were washed and co-transfected in 50 μl of final volume with CEC1 or LRIM1 promoter firefly luciferace fusion reporter constructs (Promega pGL3 backbone) and reference pAct5C-Renilla luciferase construct using Effectene Transfection Reagent (QIAGEN) following the manufacturer's instructions.

Techniques: Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Transcriptional Activation of TINF2 , a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

doi: 10.1371/journal.pone.0021333

Figure Lengend Snippet: A : The name of each TIN2 reporter construct was assigned according to the 5′-end nucleotide numbers of the promoter sequences inserted upstream of the ATG initiation codon. Basic refers to the pGL3-Basic vector. For each transfection, the firefly luciferase activity was normalized to the β-galactosidase activity expressed from a co-transfected β-galactosidase expression vector. The means from three independent experiments are shown for each construct; bars , SD. (***p<0.001). B : Finer promoter mapping of the region between −450 and −351, expressed as in panel A. C : Finer promoter mapping of the region between −148 and −24, expressed as in panel A. D : Verification of the functionality of the TINF2 promoter construct in various cell lines. For each transfection, the firefly luciferase activity was normalized to the Renilla activity expressed from the co-transfected pRL-CMV expression vector and expressed as in panel A. E : Schematic of the core TIN2 promoter construct showing putative Sp1 and NF-κB binding sites predicted using bioinformatic tools. “Drop 1” and “Drop 2” refer to the drops in luciferase reporter activity shown in panel A.

Article Snippet: Transcriptional binding site mutant plasmids ( ) were generated in the minimal promoter pGL3-P406 backbone by PCR mutagenesis using the QuickChange method (Stratagene) and appropriate primer sets (sequences available upon request).

Techniques: Construct, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Expressing, Binding Assay

Journal: PLoS ONE

Article Title: Transcriptional Activation of TINF2 , a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

doi: 10.1371/journal.pone.0021333

Figure Lengend Snippet: A : EMSA showing the ability of Sp1 to bind to its two predicted sites in vitro. Sp1 protein was in vitro translated using the rabbit reticulocyte lysate (RRL) system and incubated with end-labeled DNA oligos encompassing the putative binding sites. These complexes (lanes 3 and 9) could be competed away with unlabeled wild-type oligos (lanes 4 and 10), but not with a mutant form (lanes 5 and 11). Furthermore, the complexes could be super-shifted using an anti-Sp1 antibody (lanes 6 and 12). B : The ability of Sp1 to bind to the endogenous TINF2 promoter in vivo was shown using ChIP. The 407 base pair fragment could be amplified from a reaction including the anti-Sp1 antibody (lane 7), but not from reactions containing anti-c-Myc, normal IgG, or no antibody (lanes 4–6). C : Sp1 is the major Sp transcription factor that can activate the TINF2 promoter. Drosophila melanogaster SL2 cells were co-transfected with the minimal promoter reporter construct pGL3-P406 and various amounts of expression vectors encoding either Sp1 or Sp3. For each transfection, the firefly luciferase activity was normalized to the total protein concentration. The means from three independent experiments are shown; bars , SD.

Article Snippet: Transcriptional binding site mutant plasmids ( ) were generated in the minimal promoter pGL3-P406 backbone by PCR mutagenesis using the QuickChange method (Stratagene) and appropriate primer sets (sequences available upon request).

Techniques: In Vitro, Incubation, Labeling, Binding Assay, Mutagenesis, In Vivo, Amplification, Transfection, Construct, Expressing, Luciferase, Activity Assay, Protein Concentration

Journal: PLoS ONE

Article Title: Transcriptional Activation of TINF2 , a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

doi: 10.1371/journal.pone.0021333

Figure Lengend Snippet: A : The ability of NF-κB to bind to the endogenous TINF2 promoter in vivo was shown using ChIP. The 407 base pair fragment could be amplified from a reaction including the anti-p65 antibody (lane 5), but not from reactions containing no antibody or normal IgG (lanes 3 and 4). B : The ability of NF-κB to activate the minimal TINF2 promoter was verified by co-transfection of vectors encoding p50 and/or p65 protein and pGL3-P406 into NIH 3T3 (p50 − /p65 − ) cells. For each transfection, the firefly luciferase activity was normalized to the total protein concentration. The means from three independent experiments are shown; bars , SD. (***p<0.001).

Article Snippet: Transcriptional binding site mutant plasmids ( ) were generated in the minimal promoter pGL3-P406 backbone by PCR mutagenesis using the QuickChange method (Stratagene) and appropriate primer sets (sequences available upon request).

Techniques: In Vivo, Amplification, Cotransfection, Transfection, Luciferase, Activity Assay, Protein Concentration

Journal: PLoS ONE

Article Title: Transcriptional Activation of TINF2 , a Gene Encoding the Telomere-Associated Protein TIN2, by Sp1 and NF-κB Factors

doi: 10.1371/journal.pone.0021333

Figure Lengend Snippet: A : Mithramycin A, an Sp1 inhibitor, reduces TINF2 promoter-driven luciferase activity. 293T cells are transfected with either the promoter-less pGL3-Basic plasmid, the NFAT-responsive pNFAT-Luc plasmid, the SV40 promoter-containing pGL3-Control plasmid, or the minimal TIN2 promoter P406 plasmid and incubated with the indicated concentrations of drug for 24 hours. For each transfection, the firefly luciferase activity was normalized to Renilla luciferase activity expressed from a co-transfected Renilla luciferase expression vector. The means from three independent experiments are shown; bars , SD. (**p<0.01, ***p<0.001). B : Bay11-7082, an NF-κB inhibitor, reduces TINF2 promoter-driven luciferase reporter activity. Cells are processed and values expressed as in panel A. (***p<0.001). C : PDTC, an NF-κB inhibitor, reduces TINF2 promoter-driven luciferase reporter activity. Cells are processed and values expressed as in panel A. (***p<0.001). D . Mithramycin A and Bay11-7082 (Bay11) reduce endogenous TINF2 gene expression. The levels of TINF2 mRNA were normalized to those of the housekeeping GAPDH gene. The means from three independent experiments are shown.

Article Snippet: Transcriptional binding site mutant plasmids ( ) were generated in the minimal promoter pGL3-P406 backbone by PCR mutagenesis using the QuickChange method (Stratagene) and appropriate primer sets (sequences available upon request).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Incubation, Expressing