Fig. 2

Effect of IGF2BP1 knockout in ETV6::RUNX1 positive Reh cell line A) mRNA expression of IGF2BP1 in different leukemia cell lines (Reh, AT1, AT2, UoCB6 – ETV6-RUNX1 translocation positive cell lines and RS4;11, NALM6, THP1 – ETV6-RUNX1 translocation negative cell lines) analyzed by qRT-PCR. POLR2A and HGPRT were used as internal controls. B) Western Blot showing protein expression of IGF2BP1 in multiple cell lines. GAPDH was used as the internal control. C) Schematic for cloning of guide RNAs targeted against different exons of IGF2BP1 in pLKO5-tRFP vector D) Western blotting after knockout of IGF2BP1 in Reh Cas9-GFP cells with β-Actin as the loading control; NT (Non-targeting control) E) Cell proliferation of different IGF2BP1 KO clones as determined by MTS assay (t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) F) Effect of Prednisolone induced cytotoxicity in Reh-Cas9 cells after IGF2BP1 KO using MTS assay (IC50 ~ 1 μM for sg1/2) G) Gene Set Enrichment Analysis (GSEA): Hallmark pathways enriched after RNA-Seq of IGF2BP1 KO in Reh-Cas9 cells; The x axis represents the pre-ranked list of genes based on PC1 loadings, which segregates between genes more expressed in IGF2BP1 KO cells (left) and wild type cells (right). Segment plots (bottom) highlight the position of genes from hallmark pathways in the pre-ranked list. The vertical axis in line plots (top) represents the cumulative Enrichment Score (ES) from GSEA, and NES is the overall normalized enrichment score (with FWER=familywise error rate) for each selected pathway. Color-coded names for some genes in selected pathways are shown