The increased Lin28B expression that results from Rpl22 loss or inactivation is dependent on NF-κB activity. (A) Measurement of NF-κB activity in Rpl22-haploinsufficient primary MEF. EMSA analysis was performed using equal quantities of nuclear extract protein from primary MEFs of the indicated genotypes using both an intact (wt) and p65 binding mutant (mut) NF-κB probe. NF-κB activity was measured in untreated cells, cells pretreated with leptomycin B to trap NF-κB in the nucleus, and after TNFα stimulation (positive control). The composition of the NF-κB complexes was evaluated using supershift analysis using the indicated Abs. Effect of NF-κB inhibition on Lin28B expression. (B) Lin28B mRNA levels were quantified by real-time PCR on RNA extracted from immortalized MEFs stably expressing 2 different Rpl22 shRNAs, in which NF-κB activity had been pharmacologially inhibited by treatment with 1μM NF-κB inhibitor, IMD-350. **P < .01 for IMD-350 treated compared with control treated. (C) Rpl22 was knocked down by shRNA in primary MEF from p65 wild-type (p65+) or Rela^−/−, p65 knockout mice (p65⁻). Lin28B induction was blocked in p65 knockout cells in which Rpl22 was knocked down. Lin28B and Rpl22 mRNA levels were quantified by real-time PCR, and data are plotted as the average of 2 experiments. (D) Model of Rpl22 function in transformation. The model proposes that Rpl22 normally acts to restrain NF-κB activity by an unknown mechanism. However, when Rpl22 expression is diminished either by shRNA knockdown or mutation, NF-κB activity is increased, resulting in increased expression of Lin28B. Lin28B, in turn, promotes transformation at least in part by repressing Let-7 MiR processing, which results in derepression of oncogenic targets such as c-myc. Rpl22 is also likely to regulate additional targets that contribute to transformation.