Fig. 7: MBNL1 loss perturbs essential MLL-rearranged leukemia genes through missplicing.

a RT-PCR demonstrating the predicted increase in intron retention at alternative splice sites in DOT1L and SETD1A transcripts in MOLM13 cells after MBNL1 knockdown. For DOT1L, Actin serves as a loading control, and primer product (“Intron Inclusion Band”) only occurs when the intron is retained. Representative gel images shown, two biological replicates performed. b qRT-PCR results from RNA-immunoprecipitation assay assessing for enrichment of DOT1L and SETD1A transcripts in MBNL1-precipitated RNA. Error bars show mean ± SD. Data is from three biological replicates for MOLM-13 and two biological replicates for RS4;11. c Western blots of MOLM13 and MV4;11 cells following knockdown of MBNL1, demonstrating changes in DOT1L and SETD1A protein levels. Representative western blots shown, two biological replicates performed. SETD1A image represents same samples run and processed in parallel on separate blot. d Volcano plots representing differentially expressed genes in MOLM13 cells at 4 days (left) and 12 days (right) after knockdown. X-axis thresholds represent 1.5-fold change in expression; Y-axis threshold represents p < 0.01. Differentially expressed genes determined by empirical Bayes two-tailed moderated t-test. e Differentially expressed genes were significantly enriched positively and negatively by GSEA for corresponding gene sets related to perturbation of essential MLL leukemogenesis processes (i.e., HOXA9, upper GSEA plots; GSK3, lower plots.) Enrichment scores, p values, and false-discovery rate q values calculated as previously published⁵¹.