Journal: bioRxiv
Article Title: Centrosome architecture and m6A-dependent gating of p53 surveillance after whole-genome doubling
doi: 10.64898/2026.02.25.707964
Figure Lengend Snippet: (A) Schematic overview of the genome-wide CRISPR knockout screen in Cal51 mScarlet-MDM2 cells. Cells were transduced with the Brunello sgRNA library, subjected to puromycin selection, treated with ZM for 40 h, and sorted by FACS based on reporter fluorescence. Genomic DNA from sorted populations was processed for sgRNA amplification and Illumina-based next-generation sequencing. (B) Waterfall plot showing genome-wide ranking of genes based on MAGeCK analysis of sgRNA enrichment in the reporter-negative population. Genes are ordered by rank, and the y-axis represents the -log10-transformed MAGeCK enrichment score (robust rank aggregation score). Selected pathway components are highlighted. (C) sgRNA-level log 2 fold-change (LFC) values for selected genes identified in the CRISPR screen. Each point represents an individual sgRNA from the Brunello library targeting the indicated gene, illustrating the consistency of sgRNA behavior across hits. NTC: non-targeting control. (D) Immunoblot analysis of an isogenic panel of RPE1 cells comprising wild-type p53 cells (WT), TP53 -/- cells, or cells expressing hotspot mutant p53 alleles (R175H or R248Q) following ZM treatment. Two independent clones are shown for each TP53 -/- , R175H, and R248Q genotype. (E) Immunoblot analysis of wild-type (WT) and CRISPR-engineered Cal51 cells harboring point mutations disrupting the p53 response elements (p53REs) in the PIDD1 or MDM2 promoters, as schematized in Fig. S4A, following ZM treatment. Two independently derived clonal cell lines are shown for each genotype. (F) Dot plot of Gene Ontology (GO) Cellular Component terms enriched among the top 200 genes identified in the CRISPR screen. Dot size represents the number of genes associated with each term, and color indicates enrichment significance. (G) Jaccard similarity heatmap showing pairwise overlap between the five top-ranked enriched GO Cellular Component terms shown in (F). Each square reports the Jaccard similarity index, and colour intensity reflects similarity magnitude, highlighting extensive overlap among m6A writer-associated terms and no overlap with the centriole term.
Article Snippet: The following antibodies were used: rat monoclonal anti-Caspase-2 (a gift from Dr. Andreas Strasser, Walter and Eliza Hall Institute of Medical Research, clone 11B4, 1:1000), mouse monoclonal anti-MDM2 (Thermo Fisher Scientific, MA1-113, clone IF2, 1:1000), rabbit monoclonal anti-V5-tag (CST®, 13202, clone D3H8Q, 1:1000), rabbit polyclonal anti-p53 (CST®, 9282, 1:1000), mouse monoclonal anti-MCL1 (SCBT, sc-12756, clone 22, 1:500), rabbit polyclonal anti-CEP83 (Atlas antibodies, HPA038161, 1:1000), rabbit polyclonal anti-PARP1 (CST®, 9542, 1:1000), rabbit polyclonal anti-Caspase-3 (CST®, 9662, 1:1000), rabbit polyclonal anti-Caspase-7 (CST®, 9492, 1:1000), mouse monoclonal anti-CDC27 (BD Biosciences, 610455, clone 35/CDC27, 1:1000), mouse monoclonal anti-p21 (BD Biosciences 564262, clone 2G12, 1:1000), rabbit monoclonal anti-METTL3 (CST®, 96391, clone D2I6O, 1:1000), rabbit polyclonal anti-METTL14 (ProteinTech®, 26158-1-AP, 1:1000), rabbit polyclonal anti-CRADD/RAIDD (ProteinTech®, 10401-1-AP, 1:1000), rabbit monoclonal anti-VIRMA (CST®, 88358, clone D4N8B, 1:1000), rabbit polyclonal anti-WTAP (CST®, 56501, 1:1000), rabbit polyclonal anti-ZC3H13 (Boster Bio, A11022-1, 1:500), rabbit polyclonal anti-CBLL1 (Bethyl Laboratories®, A302-968A, 1:1000), mouse monoclonal anti-RBM15 (ProteinTech®, 66059-1-Ig, clone 4A1A4, 1:1000), mouse monoclonal anti-HSP90 (SCBT, sc-13119, clone F-8, 1:10000), rabbit monoclonal anti-GAPDH (CST®, 2118, clone 14C10, 1:10000), polyclonal goat anti-rat IgG/HRP (Thermo Fisher Scientific, 31470, 1:5000), polyclonal goat anti-rabbit IgG/HRP (Agilent, P0448, 1:5000), polyclonal rabbit anti-mouse IgG/HRP (Agilent, P0161, 1:5000).
Techniques: Genome Wide, CRISPR, Knock-Out, Transduction, Selection, Fluorescence, Amplification, Next-Generation Sequencing, Transformation Assay, Control, Western Blot, Expressing, Mutagenesis, Clone Assay, Derivative Assay