Eμ-TCL1 leukemia cells with biallelic loss of CDKN2A, CDKN2B, and TP53 proliferate spontaneously in vitro. (A) Absolute number of viable CDKN2A/2B/TP53-targeted, CDKN2A/2B-targeted, TP53-targeted, or control TCL1-355 leukemia cells at different time points in culture. A total of 5 × 106 leukemia cells of each genotype were isolated from spleens of corresponding mice and placed in culture. The number of viable cells was calculated by evaluating the ratio of the percentage of propidium iodide (PI)-negative cells out of the total number of cells. Each data point represents an average of 5 independent experiments with CDKN2A/2B/TP53-targeted and 3 independent experiments with CDKN2A/2B-targeted, TP53-targeted, and control leukemia cells. (B) May-Grünwald-Giemsa staining of control TCL1-355 leukemia cells and TCL1-355 leukemia cells with biallelic CDKN2A/2B/TP53 disruption. Scale bars, 20 μm. (C) Forward scatter (FSC) analysis of control TCL1-355 leukemia cells and TCL1-355 leukemia cells with biallelic CDKN2A/2B/TP53 disruption. (D) Indel analysis by amplicon capillary electrophoresis of CDKN2A/2B/TP53-targeted leukemia cells at day 1 and day 49 in culture. The position of the wild-type allele is indicated by a red arrow. (E) Immunoblotting analysis of TP53, CDKN1A, CDKN2A, and CDKN2B protein expression in 2 different clones of spontaneously proliferating TCL1-355 leukemia cells with biallelic CDKN2A/2B/TP53 disruption. (F) Alignment of mutant and wild-type nucleotide sequences (upper and lower panel, respectively) of the Cas9-targeted region using the Inference of CRISPR Edits (ICE) software tool. Analysis was performed on TP53/CDKN2A/2B-ko leukemia cells collected after 7 weeks in culture. (G) Analysis of the percentage of individual indels in the whole population using the ICE software tool. Wild-type allele corresponds to position 0 and is absent in all 3 panels.