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. 2017 Jul 28;6:e27155. doi: 10.7554/eLife.27155

Figure 1. The IMP2 gene is amplified and overexpressed in many cancers and drives proliferation.

Figure 1.

(A) The frequency of IMP1,2,3 gene copy number amplification in various cancers. Data from TCGA. (B) IMP1, 2 and 3 mRNA levels in various cancers. Data from TCGA. (C) IMP2 overexpression enhances proliferative rate. A vector encoding an IMP2 cDNA downstream of a doxycycline sensitive promoter was stably expressed in HCC-1419, NCI-H2029 and MEFs. Cells were treated with Doxycycline at the doses indicated and cell number was determined daily. +p<0.05, *p<0.01 vs DMSO. (D) CRISPR-mediated inactivation of the IMP2 genes slows proliferation. The Hep3b, HeLa, RD, HCC-1359, MB-231 and SNU-423 cell lines were transfected with Cas9/CRISPR and a guide RNA directed at either GFP (black) or IMP2 (red) sequences. Unselected polyclonal cell mixtures were plated in replicate and cell number was determined daily. *p<0.01 vs Imp2 CRISPR. (E) Imp2−/− MEFs proliferate more slowly than Imp2+/+ MEFs. Littermate embryos from Imp2+/− ± were harvested at e12.5–13.5 to derive Imp2+/+ and Imp2−/− MEFs. Polyclonal mixtures were plated in replicate at passage 4 and cell number was determined daily. *p<0.01 vs Imp2−/−. (F) Imp2−/− MEFs produce less medium IGF2 than Imp2+/+ MEFs. Aliquots of the medium were taken at day 3 from the MEF cultures in Figure 1E and assayed for IGF2 polypeptide. *p<0.01 vs Imp2+/+. (G) Supramaximal IGF2 does not restore the slower proliferation of Imp2−/− MEFs to that of Imp2+/+ MEFs. Imp2+/+ and Imp2−/− MEFs were plated in replicate in standard culture medium with the addition of BSA (1 ug/ml) or various amounts of human recombinant IGF2 and cell number was determined 48 hr later. +p<0.05 vs BSA.