HNRPK binding to Myc mRNA and effect of ectopic Myc expression on the leukemic phenotype of S284/353A-HNRPK-expressing 32D-BCR/ABL cells. (Ai) RT-PCR shows presence of Myc mRNA in anti-HNRPK and anti-HA immunoprecipitates from lysates of parental (lane 2) and S284/353(A/D) (lanes 3-4) HNRPK-expressing 32D-BCR/ABL cells, respectively. Lane 1 shows levels of Myc in total mRNA, whereas lane 5 represents RT-PCR of mRNA immunoprecipitated with a nonrelated (anti-Flag) antibody; (Aii) REMSA (top) and UV cross-linking (bottom) with cytoplasmic extracts of 32Dcl3, 32D-BCR/ABL, and K562 cells show binding of HNRPK to the HNRPK binding site contained in the MYC IRES element. (B) Graphs shows firefly luciferase activity after normalization with Renilla luciferase activity in 32D-BCR/ABL cells transduced with the empty pRF and with the pRMF(IRES-MYC) plasmid containing the MYC IRES element cloned in front of the firefly luciferase cDNA. (C) Effect of ectopic MYC expression on the growth factor-independent colony formation (i) and tumorigenic potential (ii) of S284/353A-HNRPK-expressing 32D-BCR/ABL cells. Inset in panel Ci shows total Myc protein levels in vector-transduced, S284/353A-HNRPK/MYC-transduced, and S284/353A-HNRPK/MYC-transduced 32D-BCR/ABL cells. (D) Effect of MYC overexpression on the growth factor-independent clonogenic potential of CD34⁺ bone marrow CML-BC patient cells ectopically expressing the S284/353A-HNRPK mutant. Primary CD34⁺ CML-BC marrow cells were transduced with the MigR1-S284/353A-HNRPK-HA construct, selected for GFP⁺, and infected again with the MSCV-puro-MYC retrovirus. In panels B-D, bars represent mean ± SE of values from 3 different experiments.