Early erythroid development is impaired in RPS19-deficient BM cells. (A) Proliferation in erythroid differentiation medium (IMDM containing FCS [30%], hydrocortisone [10^-6 M], 2-ME [10^-4 M], Epo [10 U/mL], IL-3 [0.01 ng/mL], and IL-6 [0.001 ng/mL]) is decreased in RPS19-silenced cells. At day 7 cell counts were reduced in LV-TH-RPS19-C- and LV-TH-RPS19-B-transduced cells with a statistical significance. (B) The fraction of hemoglobin-containing cells, microscopically scored by DAF staining, was reduced in LV-TH-RPS19-B- and LV-TH-RPS19-C-transduced cells at day 7. (C) The stage of erythroid differentiation was determined in GFP-positive cells from day 14 of erythroid liquid culture. The FACS profile using cell surface markers CD71 and Glycophorin A distinguish between myeloid cells CD71^lo GlyA^lo/- (R1), erythroid-committed progenitors CD71⁺ GlyA^int (R2), and more mature erythroid cells GlyA^hi (R3). Representative FACS plots from healthy CD34⁺ BM cells transduced with the vectors LV-TH-RPS19-Scr, -A, -B, and -C and CD34⁺ BM cells from an RPS19-deficient DBA patient transduced with the LV-TH-RPS19-Scr vector are shown. (D) Cytospin slides of normal cells from the 3 gates. (E) The fraction of GFP-positive cells in erythroid culture that has not committed to the erythroid lineage (R1) increases in RPS19-silenced cells. The fraction of GFP-positive committed erythroid progenitors (R2) and mature erythroid cells (R3) decrease in RPS19-silenced cell cultures. For panels A, B, and E, the means ± SEMs for 5 independent experiments are shown compared with LV-TH-RPS19-Scr-transduced cells. The experiments with DBA patient BM cells are repeated twice. ^*P < .05, ^**P < .01 compared with LV-TH-RPS19-Scr-transduced cells.