Lin28B is necessary for the enhancement of growth and transformation by Rpl22 inactivation. (A) Effect of knockdown of Lin28B on the growth of immortalized Rpl22^−/− MEFs. Immortalized MEFs of the indicated genotypes were transduced with control or Lin28B shRNA. The effect on expression of Lin28B was assessed by immunoblotting. Growth of triplicate wells of MEFs stably expressing control shRNA or Lin28B shRNA was determined by counting and then plotted as the mean cell number ± SD *P < .05, for Rpl22^−/− compared with Rpl22^−/− in which Lin28B was knocked down. (B) Effect on growth of reintroducing Rpl22 into Rpl22^−/− MEF. Immortalized Rpl22^−/− MEFs were transfected with Rpl22 or empty vector (EV) control, after which we assessed cell growth by counting triplicate wells and depicting the mean ± SD graphically. The expression of Lin28B, c-myc, Rpl22, and GAPDH (loading control) were evaluated by immunoblotting. (C) Dependence of soft agar colony formation on expression of Lin28B. Primary MEFs of the indicated genotypes were transduced with Lin28B shRNA followed by E1A and Ras, and then plated in triplicate in soft agar as Figure 3B. Representative images were captured with a Digital Slight DS-Fi1 camera and NIS Element AR3.0 imaging software at 1× with 3× zoom (×30 total magnification) using a Nikon SMZ1500 stereomicroscope and are shown in the top panels. The mean colony number ± SD is represented graphically beneath. **P < .005 for colonies in Rpl22^+/− and −/− relative to Rpl22^+/+. (D) Lin28B expression in Rpl22-haploinsufficient thymic lymphomas. Explanted thymic lymphomas from MyrAkt2;Rpl22^+/+ and MyrAkt2;Rpl22^−/− mice were evaluated for Lin28B and c-myc expression by immunoblotting. GAPDH served as a loading control. (E) Lin28B mRNA levels in RPL22^+/+ and Rpl22^+/− human T-ALL lines. Lin28B mRNA levels in the indicated T-ALL cell lines were quantified by real-time PCR. Data are presented as Log2 value relative to Jurkat cells (control).