Silencing of MLL-AF6 in t(6;11)(q27;q23) rearranged cells restores AF6 in the cytoplasm. (A) Western blot (WB) analysis revealed decreased levels of MLL-AF6 after silencing (siMLL-AF6) compared with negative controls (scRNA) in both, ML2 and SHI-1. Anti-ACTIN was used as endogenous control. (B) The siMLL-AF6 cells restored AF6 protein in the cytoplasm. Immunofluorescence shows colocalization of AF6 (red) and RAS (green) in SHI-1 after MLL-AF6 silencing (merged signals, yellow, nuclei blue, ×60 zoom). In the negative control (scRNA), the punctuate pattern of AF6 nuclear localization is visible (red AF6, nuclei blue, ×60 zoom). (C) The siMLL-AF6 cells restored AF6 protein in the cytoplasm. Immunofluorescence shows colocalization of AF6 (red) and RAS (green) in primary t(6;11)-AML after MLL-AF6 silencing (merged signals yellow, nuclei blue, ×60 zoom). In the negative control (scRNA), the punctuate pattern of AF6 nuclear localization is visible (red AF6, nuclei blue, ×60 zoom). (D) Active RAS-GTP levels in ML2 cell line silenced for the chimera showed a decreased activity of RAS (0.47) compared with scRNA. (E) Luciferase (LUC) activity of ML2 transfected with a pFOS (FBJ murine osteosarcoma viral oncogene homolog) WT-GL3 plasmid and siMLL-AF6 show a reduction of LUC activity compared with scRNA. Introduction of both siRNA for MLL-AF6 and AF6 show a rescue of LUC activity in ML2. (F) WB of p-ERK1/2 and total ERK in ML2 silenced for MLL-AF6 and in double silencing of MLL-AF6 and AF6 compared with scRNA. (Right) Histogram represents the ratio between p-ERK and total ERK: a reduction of p-ERK is visible after MLL-AF6 silencing, and a rescue of p-ERK is documented when also AF6 was silenced. *P > .05.