MUC1 is critical in the expansion of MDSCs. Stable MOLM-14 and THP-1 AML cell lines silenced for the expression of MUC1 protein were generated by lentiviral transduction of shRNA or control vector. (A) Lysates were prepared and cells analyzed for MUC1 expression using Western blotting. MOLM-14 cells were silenced for MUC1 expression using CRISPR/Cas9 technology to validate the silencing. (B) Lysates were prepared and cells analyzed for MUC1 expression using Western blotting using β-actin as a loading control. Healthy PBMCs and irradiated, fluorescently labeled MUC1-silenced and control AML cells were cocultured for 5 days at a ratio of 100:1 (PBMC:AML). After coculture, cells were analyzed with flow cytometry, and fluorescently labeled blast cells were excluded; CD11b⁺/HLA-DR⁻/CD33⁺ MDSCs were quantified as a percentage of immature CD11b⁺/HLA-DR⁻ myeloid cells. Summary of three independent experiments for (C) MUC1- silenced MOLM-14 and THP-1 AML cells and (D) CRISPR/Cas9 MUC1-silenced MOLM-14 cells are shown (*P < .05). CRISPR, clustered regularly interspaced short palindromic repeats; IB, immunoblot; WT, wild-type.