Figure 1. RNAi screen identifies Eed and Suz12 as unique requirements for growth of MLL-AF9;Nras^G12D leukemia.

(A) Scatter-plot comparison of the relative growth inhibition conferred by LMN-shRNAs in MLL-AF9;Nras^G12D leukemia and 32D myeloblasts. All shRNAs evaluated were identified from a pooled negative-selection shRNA screen reported previously (25). MLL-AF9;Nras^G12D leukemia or 32D myeloblasts were transduced with individual LMN-shRNA vectors (MSCV-miR30-shRNA-PGK-NeoR-IRES-GFP), followed by measurement of the GFP-percentage at day 2 and day 12 post-infection using a Guava Easycyte (Millipore). Growth inhibition was calculated as the ratio of the GFP% measured at day 2 to day 12 of partially transduced cell populations. Since leukemia and 32D cells grow at comparable rates in vitro (Supplementary Figure 1), relative GFP-depletion is a suitable assay for comparing growth effects in each line. Control shRNAs are indicated with white circles. Box indicates shRNAs with leukemia-specific growth inhibition. (B–F) Relative growth inhibition conferred by indicated LMN-shRNAs in EML, G1E, leukemia, and 32D cell lines, calculated as in (A) (n = 3). (G–I) Quantitative reverse transcription PCR measuring knockdown efficiency in 32D myeloblasts cells following transduction with LMN-shRNAs and selection with G418. Measurements were normalized to Gapdh, with the relative mRNA level in the cells with control Ren shRNA set to 1 (n = 3). (J) Relative change double-transduced cell percentage following co-transduction with indicated LMN-shRNAs linked to either GFP or mCherry reporters. The results were normalized to the GFP+/mCherry+ percentage measured at day 1, set to 1 (n = 3). (K, L) H3K27me3 Western blotting of acid extracted histones prepared from 32D cells transduced with the indicated LMN-shRNA following G418 selection. The levels of total histone H3 serve as a loading control. A representative experiment of three replicates is shown. All error bars represent s.e.m.