RPS19-deficient cells show increased expression of TNF-α. CD34⁺ hematopoietic progenitor cells were transduced with lentivirus carrying shRNA against RPS19 or Luc control, sorted for GFP⁺ cells at 72 hours, and analyzed 5 days after infection. (A) TNF-α mRNA and (B) protein levels are increased in the media from RPS19 deficient cells, compared with control, as measured by enzyme-linked immunosorbent assay (ELISA). Each symbol on the graph represents a separate measurement [n = 4 for Luc and RPS19 (sh3); n = 5 for RPS19 (sh1); n = 6 for RPS19 (sh2)]. (C) TNF-α is predominantly expressed in the CD71^– (nonerythroid) fraction of cord blood cells in culture. (D) Cells were analyzed for CD71 and TNFR1 expression by flow cytometry. TNFR1 expression increased on the surface of RPS19-deficient CD71⁺ cells compared with Luc control but did not change in the CD71^– population. (E) Addition of 100 ng TNF-α to CD34⁺ cord blood cells results in decreased GATA1 and increased p21 expression after 24 hours. This effect is rescued by addition of 10 μg etanercept (Eta). Data are representative of 2 independent experiments. *P < .05; **P < .01; ***P < .001.