In vitro and in vivo alterations of SF3B1 gene lead to an RARS phenotype. (A) K562 cells were transfected with shRNA constructs. After 48 hours of puromycin selection, RT-PCR assay was performed using random oligos for endogenous U2 (1-7) and U12 (1-3) introns (Table 1). The ratio of spliced to unspliced pre-mRNA for U2- and U12-dependent introns is shown. Average reaches 0.42 and 1.89 for U2-introns and U12-introns, respectively. Data are mean ± SD, calculated from 2 independent experiments. K562 transfected with vector only was used as control, and the ratio of spliced to unspliced pre-mRNA was set to 1. (B) Human colony-forming unit cell assay was performed on BM cells derived from 4 healthy persons. Cells were treated with different doses of meayamycin (2, 10, and 50nM). Representative pictures at 2 and 50nM are shown. Colonies were harvested after 2 weeks, spotted on cytospin slides, and subjected to Prussian blue staining. (C) Prussian blue staining of BM aspirates shows numerous RSs in Sf3b1^+/− compared with C57BL/6 mice. Closed arrowheads indicate perinuclear RS. BM cytospin slides were kindly provided by Dr H. Koseki from Japan. Slides were analyzed using an Olympus system microscope (model BX41; objective lens, ×100; camera, SPOT Idea Model No 28.2). Images were acquired using a SPOT Imaging software by Diagnostic Instruments Inc (www.Diaginc.com).