RPL5^+/p.R23X iPSCs exhibit ribosomal defects that are rescued by gene correction. (A) Western blot for RPL5 protein in WT1 and RPL5^+/p.R23X iPSCs (first 2 lanes). Gene-corrected and control sublines were generated by introducing WT RPL5 or GFP cDNA, respectively, into the AAVS1 locus of RPL5^+/p.R23X iPSCs (lanes 3 and 4). Ratio of RPL5:actin analyzed by densitometry is shown relative to the WT1 clone, which is assigned an arbitrary value of 1.0. (B) Polysome profiling of RPL5^+/p.R23X iPSCs and sublines with GFP or WT RPL5 cDNAs integrated into the AAVS1 locus. A polysome profile of a WT iPSC line is shown in Figure 1B. Arrow shows direction of the sucrose gradient from less to more dense. (C) Summary of 3 polysome profiling experiments showing increased ratio of 40S:60S ribosomal subunits in RPL5-haploinsufficient iPSCs (lanes 2 and 3), compared with WT (lane 1) or RPL5-rescued RPL5^+/p.R23X iPSCs (lane 4). *P < .05. (D) Northern blot analysis with the ITS2 probes (see Figure 1D) showing accumulation of 12S pre-rRNA molecules in RPL5^+/p.R23X iPSCs (designated as “–” in the last lane of the left panel) compared with WT cells. This defect is ameliorated in RPL5^+/p.R23X iPSCs expressing WT RPL5 cDNA, but not in those expressing GFP cDNA (right). The ratios of 17S:12S rRNAs determined by densitometry scanning are shown at the bottom.