Fig. 6

Positive feedback between ETV6::RUNX1 and IGF2BP1 A) RT-PCR of both Input and Immunoprecipitated RNA fraction for different known targets of IGF2BP1: ETV6::RUNX1 junction (298 bp), MYC CRD region (357 bp) and 5’ UTR of ETV6 (154 bp) followed by agarose gel electrophoresis. This reveals ETV6::RUNX1 and 5’-ETV6 as targets; β-actin 3’ UTR (184 bp) was used as a positive control B) Dual luciferase assay in 293T cells reveals stabilization of the ETV6::RUNX1 junction by IGF2BP1 C) Effect of IGF2BP1 KO on ETV6::RUNX1 fusion transcript stability as determined by qRT-PCR D) Cell proliferation assay (MTS) shows reduced proliferation of ETV6::RUNX1 KO Reh-Cas9 cells E) Reversal of prednisolone resistance after ETV6::RUNX1 KO in Reh-Cas9 cells determined after 72 hours using MTS assay (IC50=1.6/2/3.5 μM for sg1/2/3 respectively) (t-test; p * <0.05, ** <0.01, *** <0.005, **** <0.0001) F) Real Time PCR shows dose dependent increase in IGF2BP1 levels after overexpression of ETV6::RUNX1 fusion transcript in 7OZ/3