pb halo ires egfp plasmid  (Addgene inc)

Journal: eLife

Article Title: Δ133p53α and Δ160p53α isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.7554/eLife.106469

Figure Lengend Snippet: ( A–D ) H1299 cells were transfected with luciferase reporter plasmids driven by p21 ( A ), MDM2 ( B ), BAX ( C ), and PUMA ( D ) promoters, along with vectors expressing FLp53, Δ133p53, or Δ160p53. To evaluate the influence of the isoforms on FLp53’s transactivation capability, co-expression was performed at ratios of 1:1, 1:5, and 1:10 relative to FLp53. Basal promoter activity was determined by transfecting cells with the empty vector, pcDNA3.1. Relative promoter activity is shown after normalization to the pcDNA3.1-treated sample activity. Data represent mean values ± standard deviation (SD) (n=3). *p<0.05; **p<0.01 (Student’s t-test). ( E–H ) The transcriptional activity of FLp53 on the p21 ( E ), MDM2 ( F ), BAX ( G ), and PUMA ( H ) promoters was inhibited by Δ133p53 and Δ160p53. Inhibition curve fitting was performed using the exponential function ExpDec1 in Origin 2018 software. Figure 4—source data 1. Luciferase reporter assay data corresponding to . Figure 4—source data 2. Data corresponding to , showing the inhibition of FLp53’s transcriptional activity by Δ133p53 and Δ160p53.

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (RRID: Addgene_16451 ) , pGL3-MDM2-Luc (RRID: Addgene_32369 ) , and PUMA Frag1-Luc (RRID: Addgene_16591 ) ( ) were obtained from Addgene.

Techniques: Transfection, Luciferase, Expressing, Activity Assay, Plasmid Preparation, Standard Deviation, Inhibition, Software, Reporter Assay

Journal: eLife

Article Title: Δ133p53α and Δ160p53α isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.7554/eLife.106469

Figure Lengend Snippet: ( A ) Schematic representation of the promoters of p53 target genes (p21, MDM2, BAX, and PUMA). An arrow denotes the transcription start site (TSS). The DNA sequences of p53 response elements (REs) are shown, with uppercase and lowercase letters indicating matched and mismatched bases, respectively, in relation to the canonical p53 RE sequence (RRRCWWGYYY). Paired arrows highlight the regions subjected to quantitative PCR (qPCR) amplification. Specifically, the distal (5') p53 RE within the p21 gene promoter was analyzed. ( B ) Relative DNA binding of the FLp53-FLAG, Δ133p53-FLAG, and Δ160p53-FLAG proteins to p53 target genes (p21, MDM2, BAX, and PUMA) in H1299 cells. ( C ) Relative DNA binding of the FLp53-FLAG protein to the p53 target gene promoters in the presence of the V5-tagged protein Δ133p53 or Δ160p53 at a 1:1 ratio. Chromatin immunoprecipitation (ChIP)-qPCR assay data are shown as relative enrichment of promoter sequences of the target genes after normalization to the control, pcDNA3.1 plasmid transfected cells. Data are represented as the mean of technical triplicates ± standard deviation (SD), **p<0.01 (Student’s t-test). Figure 3—source data 1. Chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) assay data corresponding to .

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (RRID: Addgene_16451 ) , pGL3-MDM2-Luc (RRID: Addgene_32369 ) , and PUMA Frag1-Luc (RRID: Addgene_16591 ) ( ) were obtained from Addgene.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Amplification, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Control, Plasmid Preparation, Transfection, Standard Deviation

Journal: eLife

Article Title: Δ133p53α and Δ160p53α isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.7554/eLife.106469

Figure Lengend Snippet: ( A–D ) H1299 cells were transfected with luciferase reporter plasmids driven by p21 ( A ), MDM2 ( B ), BAX ( C ), and PUMA ( D ) promoters, along with vectors expressing FLp53, Δ133p53, or Δ160p53. To evaluate the influence of the isoforms on FLp53’s transactivation capability, co-expression was performed at ratios of 1:1, 1:5, and 1:10 relative to FLp53. Basal promoter activity was determined by transfecting cells with the empty vector, pcDNA3.1. Relative promoter activity is shown after normalization to the pcDNA3.1-treated sample activity. Data represent mean values ± standard deviation (SD) (n=3). *p<0.05; **p<0.01 (Student’s t-test). ( E–H ) The transcriptional activity of FLp53 on the p21 ( E ), MDM2 ( F ), BAX ( G ), and PUMA ( H ) promoters was inhibited by Δ133p53 and Δ160p53. Inhibition curve fitting was performed using the exponential function ExpDec1 in Origin 2018 software. Figure 4—source data 1. Luciferase reporter assay data corresponding to . Figure 4—source data 2. Data corresponding to , showing the inhibition of FLp53’s transcriptional activity by Δ133p53 and Δ160p53.

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (RRID: Addgene_16451 ) , pGL3-MDM2-Luc (RRID: Addgene_32369 ) , and PUMA Frag1-Luc (RRID: Addgene_16591 ) ( ) were obtained from Addgene.

Techniques: Transfection, Luciferase, Expressing, Activity Assay, Plasmid Preparation, Standard Deviation, Inhibition, Software, Reporter Assay

Journal: eLife

Article Title: Δ133p53α and Δ160p53α isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.7554/eLife.106469

Figure Lengend Snippet: ( A ) Mathematical modeling of tetramer formation probabilities . The probabilities (p) of tetramer formation involving different combinations of FLp53(FL) and its isoforms (I) Δ133p53 or Δ160p53 are expressed. The ratio r is defined as the concentration of Δ133p53 or Δ160p53 relative to the concentration of FLp53. ( B–E ) Prediction of isoform/FLp53 hetero-tetramer formation based on the data from . The promoter activity of p53 target genes p21 ( B ), MDM2 ( C ), BAX ( D ), and PUMA ( E ) was shown. For PUMA, promoter activities are normalized to the activity when Δ133p53 and FLp53 are co-transfected at a 1:1 ratio. The predicted inhibition curves (black dots) are derived from the tetramer formation probabilities as shown in panel A. We hypothesize that the transcription factor is fully active only when it forms a tetramer consisting of 4FL; then, the final activity A equals P 4FL . Thus, A 4FL = P 4FL ; A 3FL = P 4FL + P 3FL ; A 2FL = P 4FL + P 3FL + P 2FL ; A 1FL = P 4FL + P 3FL + P 2FL + P 1FL . The experimental inhibition curves with Δ133p53 (red) or Δ160p53 (blue) were all above the theoretical inhibition curves for two isoform molecules per tetramer. Our analyses indicate that the Δ133p53 or Δ160p53 isoforms can exert an inhibitory effect on FLp53 function only when present in the tetramer in higher proportion than FLp53.

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (RRID: Addgene_16451 ) , pGL3-MDM2-Luc (RRID: Addgene_32369 ) , and PUMA Frag1-Luc (RRID: Addgene_16591 ) ( ) were obtained from Addgene.

Techniques: Concentration Assay, Activity Assay, Transfection, Inhibition, Derivative Assay