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. Author manuscript; available in PMC: 2018 Nov 5.
Published in final edited form as: Nature. 2017 Nov 27;552(7683):126–131. doi: 10.1038/nature24678

Figure 4. Negative effect of METTL3 depletion on the translation efficiency of genes necessary for AML growth.

Figure 4

a) Western blot showing METTL3, SP1, SP2 and ACTIN protein levels in MOLM13 cells infected with METTL3-specific or CTRL TET-inducible shRNAs 8 days after doxycycline treatment. Two independent biological replicates are shown. For gel source data see Supplementary Information. b) SP1 and SP2 mRNAs in each ribosome fraction were quantified through qPCR and plotted as a percentage of the total. Data are from two independent polysome-profiling experiments. Mean ±SEM are shown..c) Schematic representation of the engineered reporter system. d) Firefly luciferase activity from UAS or scrambled (SCR) sequence carrying plasmid in presence of GAL4 either alone or fused with METTL3 wild type (CD) or inactive (CD DW/AA) catalytic domain (*p<0.05; t-test). The mean +S.D. of three independent transfections is shown, for two different cell lines (HT-29 and FADU) e) Proliferation assay of MOLM13 cells infected with METTL3-specific or CTRL TET-inducible shRNAs and with an SP1 expression vector between day 3 and day 6 after doxycycline treatment. Mean+S.D. of three independent replicates is shown. f) Competitive co-culture assay showing negative selection of BFP+ human tumour cell lines upon targeting of METTL3 or SP1 by CRISPR-Cas9. Results were normalized to day 4 for each gRNA. Mean+S.D. of two independent infections is shown.