pgl3 mdm2 luc  (Addgene inc)

Journal: bioRxiv

Article Title: Δ133p53 and Δ160p53 isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.1101/2024.07.23.604790

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. 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).

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (Plasmid #16451) , pGL3-MDM2-Luc (Plasmid #32365) , and PUMA Frag1-Luc (Plasmid #16591) were obtained from Addgene.

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

Journal: bioRxiv

Article Title: Δ133p53 and Δ160p53 isoforms of the tumor suppressor protein p53 exert dominant-negative effect primarily by co-aggregation

doi: 10.1101/2024.07.23.604790

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.

Article Snippet: Luciferase reporter plasmids, WWP/p21-Luc (Plasmid #16451) , pGL3-MDM2-Luc (Plasmid #32365) , and PUMA Frag1-Luc (Plasmid #16591) were obtained from Addgene.

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