MUC1 promotes c-myc expression in EVs, which leads to upregulation of cyclin D2 and E1 in cocultured MDSCs. Stably transduced cell lines silenced for the expression of MUC1 protein were generated by lentiviral transduction of shRNA or CRISPR/Cas9 technology. Control shRNA or wild-type cells were used as controls. Lysates were prepared, and MUC1 and c-myc expression was assessed using Western blot analysis (A) in AMLs cells and (B) in isolated secreted EVs generated from MOLM-14 and THP-1 cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and CD63 were used as loading controls. CD11b⁺/HLA-DR⁻/CD33⁺ MDSCs were isolated from healthy donor PBMCs and cultured for 48 hours with EVs isolated from the culture medium of THP-1 cells. (C) PBMCs were lysed and subjected to immunoblot for c-myc and cyclins D2 and E1. GAPDH was used as a loading control. RNA was isolated from MUC1-silenced MOLM-14 and THP-1 cells and subjected to qPCR with primers against c-myc. (D) The c-myc expression in MUC1-silenced MOLM-14 and THP-1 is quantified in relation to control cells; a summary of three experiments is shown.