(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.