Figure 2
Figure 2. Dexamethasone and lenalidomide increase the production of erythroid cells from CD34+ cells expressing RPS19 or RPS14 shRNAs without increasing apoptosis. In panels A and D, a Western blot shows the decreased level of protein with RPS19 knockdown (56.7% knockdown by qRT-PCR) and with RPS14 knockdown (61.9% knockdown by qRT-PCR). The absolute number of erythroid cells after 10 days in liquid culture was determined by multiplying the number of cells counted per well by the percentage of those cells expressing any erythroid markers (CD71, glycophorin A) as assayed by flow cytometry. Data are presented as relative numbers to eliminate differences caused by cell number and infection rate. The effects of dexamethasone (Dex), lenalidomide (Len), and control on cells infected with a RPS19 shRNA are shown in panel B. The effects of Dex, Len, and control on cells infected with a RPS14 shRNA are shown in panel E. No increase in annexin staining was noted in CD34+ cells expressing RPS19 or RPS14 shRNAs and treated with either Dex or Len compared with vehicle control, as shown in panels C and F. The experiments were performed in triplicate and the entire experiment was repeated with similar results with an independent shRNA (supplemental Figure 5). A 2-tailed Student t test was used. **P ≤ .01; *P ≤ .05.

Dexamethasone and lenalidomide increase the production of erythroid cells from CD34+ cells expressing RPS19 or RPS14 shRNAs without increasing apoptosis. In panels A and D, a Western blot shows the decreased level of protein with RPS19 knockdown (56.7% knockdown by qRT-PCR) and with RPS14 knockdown (61.9% knockdown by qRT-PCR). The absolute number of erythroid cells after 10 days in liquid culture was determined by multiplying the number of cells counted per well by the percentage of those cells expressing any erythroid markers (CD71, glycophorin A) as assayed by flow cytometry. Data are presented as relative numbers to eliminate differences caused by cell number and infection rate. The effects of dexamethasone (Dex), lenalidomide (Len), and control on cells infected with a RPS19 shRNA are shown in panel B. The effects of Dex, Len, and control on cells infected with a RPS14 shRNA are shown in panel E. No increase in annexin staining was noted in CD34+ cells expressing RPS19 or RPS14 shRNAs and treated with either Dex or Len compared with vehicle control, as shown in panels C and F. The experiments were performed in triplicate and the entire experiment was repeated with similar results with an independent shRNA (supplemental Figure 5). A 2-tailed Student t test was used. **P ≤ .01; *P ≤ .05.

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