Figure 4.

(A) MOLM13, MOLM14, OCIAMl2, OCIAML3, and GDM cell lines were infected with constitutive lentivirus encoding WT1 transcripts or an empty control with a red fluorescent protein (Dsred). The competitive growth graphs depict the percentage changes of transduced cells (Dsred+%) over 11 days in culture measured by flow cytometry. (B) MOLM13, MOLM14, OCIAMl2, OCIAML3, and GDM cell lines were infected with Dox inducible lentivirus encoding sWT1+/−. The dot plots depict the percentage changes of transduced cells (GFP%) at day 6 in the presence or absence of Dox. Significance was determined by a Wilcoxon matched-pairs signed rank test (*p < 0.05). (C) Representative cell cycle histograms for OCIAML3 cells expressing sWT1+/− or sWT1−/− (Dsred+) or non-transduced control cells (Dsred−). (D) The bar graphs show the percentages of cells in different cell cycle stages, calculated by the FlowJo Watson Pragmatic Algorithm from three cell lines (MOLM13, OCIAML2, and OCIAML3). Statistical analyses were determined by paired two-tailed t tests. (E) The immunoblot image shows p53 protein levels in cells expressing either a sgRNA targeting p53 (sgTP53) or a non-specific control sgRNA (sgNT). Vinculin and Actin served as loading controls. (F) OCIAML2 and OCIAML3 cells transduced with sgRNAs targeting TP53 were subsequently transduced with sWT1+/− DsRed plasmid following puromycin selection. Competitive growth curves show the percentage changes in the sWT1+/− expressing population (DsRed+) over time. (G) GDM and MOLM14 cells were treated with increasing doses of the MDM2 inhibitor idasanutlin for one month, generating idasanutlin-resistant cell lines that were found to harbor TP53 mutations through exome sequence analysis (paper in revision). These cells were subsequently transduced with sWT1+/− DsRed plasmid. Competitive growth curves depict percentage changes of sWT1+/− expressing cells (Dsred+%) on these idasanutlin-resistant TP53 mutant cell lines over time. (H) Bar graphs show a significant reduction in colony numbers of mouse hematopoietic stem and progenitor cells (HSPCs) expressing sWT1+/− compared to empty vector controls, both in M3434 pancytokine and M3435 myeloid-enriched methylcellulose media. Statistical analyses were determined by two-tailed t tests.