Differential gain-of-function activity of three p53 hotspot mutants in vivo

To identify mutant p53 gain-of-function, primary murine osteosarcomas expressing p53 heterozygous mutants were compared to p53 heterozygous tumors. Transcriptomes regulated by different p53 hotspots were used to identify their mechanisms of action. Validation was done in cell line expressing mutant p53, to confirm its binding to transcription factors Stat3 and Egr1. Overall design: Primary murine osteosarcomas from all genotypes were collected during necropsy, snap frozen using liquid nitrogen, crushed, and used for RNA-isolation and subsequent sequencing. QC was performed using FastQC. The reads were aligned to GRCm38 reference genome using STAR, and DEGs were identified using DESeq2, and further used for pathway analyses using IPA. Differentially upregulated genes were used to perform promoter analysis to identify enriched motifs of transcription factors using Enrichr, oPOSSUM, and IPA. Cell lines were established from primary murine osteosarcomas at the time of necropsy, and siRNA against p53 was used to knockdown mutant p53 in cell line 14.