MEF2D is specifically required for MLL-rearranged AML. (A-D) Competitive proliferation assay showing that MEF2D is required for cell proliferation in MLL-r AML cells (left, green text) but not in non-MLL-r cells (right, black text). Cells were infected with indicated guide RNA, which is linked with an mCherry gene. The mCherry positive percentage was normalized to the day 3 measurement. Two independent guide RNAs targeting MEF2D exon 4 (e4.1; A) and exon 5 (e5.1; B) were used. An sgRNA targeting the pan-essential genes RPS19 (C) was used as a positive control and an sgRNA targeting the Luciferase gene sgLuc (D) was used as a negative control. (E) Western blot analysis showing CRIPSR/Cas9-mediated MEF2D gene knockout in 4 MLL-r AML cells (green) and 4 non-MLL-r AML cells (black). Two independent guide RNAs targeting MEF2D exon 4 (e4.1) and exon 5 (e5.1) were used. GAPDH serves as a loading control. (F) Comparison of MEF2D genomic and cDNA sequences at sgRNA (e2.1) recognition sites. Dots (•) indicate mismatches. PAM, protospacer-adjacent motif. (G) Western blot analysis showing MEF2D protein levels in indicated samples. Empty, empty vector; OE, overexpression. (H) Competitive proliferation assay in indicated MOLM-13 Cas9-expressing cell lines showing the effects upon MEF2D loss can be rescued by exogenous expression of MEF2D.