Fig. 7
HOXA9 partially restores the gene transcriptional defect in RBM5 null leukemia cells. a Integrated scatter plot analysis for all genes (P < 0.05) from the RNA-seq dataset by comparing HOXA9 vs. RBM5 KO and RBM5 KO vs. CTRL in MOLM13 cells (left panel) (n = 17,079). The graph at the right shows the differentially expressed genes (DEG) in RBM5 KO vs. CTRL (n = 174). False discovery rate [FDR] < 0.05. Pearson r and P-value are calculated by correlation. b Heat map of RNA-seq analysis shows the decreased genes after RBM5 loss respectively in CTRL, RBM5 KO, and RBM5 KO with HOXA9 overexpression groups. c Enrichment of target genes involved in MLL signature genes, NUP98-HOXA9 and FLT3 targets for the increased genes in RBM5 KO cells with HOXA9 overexpression relative to control, as shown by GSEA. d. Real-time-qPCR analysis was performed on samples of CTRL, RBM5 KO (R5 KO), and RBM5 KO MOLM13 cells with HOXA9 cDNA transduction (R5 KO + HOXA9) to validate the expression of RBM5, HOXA9, FLT3, PIM1, and DDIT4 genes. Data shown are means ± SEM from three independent experiments. ***P < 0.001, ****P < 0.0001, unpaired Student’s t-test. e Western blot of HOXA9 and FLT3 were performed on samples of CTRL, R5 KO, and RBM5 KO MOLM13 cells with HOXA9 cDNA transduction (R5 KO + HOXA9). The β-ACTIN was used as a reference. f Immunoblotting was conducted by infecting OCIAML2 cells overexpressing ectopic Venus empty vector and FLT3 cDNA. The β-ACTIN was used as a reference. g Rescued competitive proliferation assay was conducted by infecting OCIAML2-Cas9 cells overexpressing ectopic RBM5-wild-type cDNA (WT), RBM5-sgRNA1-resistant mutant cDNA (PAM-MUT), and FLT3 cDNA (linked to Venus reporter) with lentiviral-sgRNAs against non-target (sgNT) and RBM5 (RBM5-sg1) at about 50% efficiency (all monitored by Venus reporter). Data shown are means ± SEM from three independent experiments. **P < 0.01, unpaired Student’s t-test. h Scatter plot analysis comparing RBM5 mRNA levels and FLT3 mRNA levels across leukemia cell lines (data was obtained from the DepMap). P-value is calculated by linear regression