Rpl22 haploinsufficiency and deficiency promote growth and transformation in cell models in vitro. (A) Effect of Rpl22 inactivation on growth of primary MEFs. Primary Rpl22^+/+, +/−, and −/− MEFs were seeded in triplicate, cultured in 3% O₂ at 37°C, and counted at the indicated intervals for 8 days. Mean cell number ± SD at each time point is represented graphically. Results are representative of 3 independent experiments. (B) Effect of Rpl22 inactivation on transformation of primary MEFs. Primary Rpl22^+/+, +/−, and −/− MEFs were transduced with oncogenes E1A and H-RasV12, followed by drug selection for 1 week, and plating in 0.7% agar. After 3 weeks, colonies were stained with crystal violet and enumerated. Images of representative wells were captured using an EPSON Perfection V700 Photo scanner and are depicted in the top panels. Mean colony number per well ± SD for each genotype is represented graphically in the bottom panel. **P < .005. Data are representative of 3 independent experiments performed in triplicate. (C) Knockdown of Rpl22 expression in immortalized MEFs. Immortalized Rpl22^+/+ MEFs were transduced with control or Rpl22 shRNA constructs, after which the effect on Rpl22 mRNA and protein expression was evaluated by real-time PCR (top) and immunoblotting (bottom). (D-E) Evaluation of immortalized MEF growth and transformation after Rpl22 knockdown. MEFs stably expressing control or 2 Rpl22 shRNA constructs were transformed by oncogenic H-RasV12, after which their growth rate was assessed by counting (panel D; *P > .05 vs control shRNA) and their transformation by colony formation in soft agar (E) as in panel B.