EPO induces the expression of TNF-α in hematopoietic cell lines.

All panels represent RNAse protection analysis of total RNA isolated from the cell lines indicated. Arrows on left indicate the presence of protected fragments for lymphotoxin β (LTβ), TNF-α, interleukin-6 (IL-6), TGF-β2 (TGF-β), interferon gamma (IFN-γ), interferon-β (IFN-β), macrophage inhibitory factor-1 (MIF-1), and housekeeping genes L32 and glyceraldehyde phosphate dehydrogenase (GADPH). P indicates undigested probe. (A) HCD57 cells were deprived of EPO for 18 hours and then stimulated with nothing (lane 1) or EPO (lanes 2-7) for the times indicated. Mouse control RNA (lane 8) and yeast RNA (lane 9) were used as positive and negative controls for the RNAse protection, respectively. Lines on right of panel indicate location of undigested probe; arrows on left indicate protected fragments indicative of expression of factors and housekeeping genes. (B) HCD57 cells were deprived of EPO overnight and then stimulated with nothing (lane 1), EPO (lanes 2-4), SCF (lanes 5, 6), or TNF-α (lanes 7, 8) for the times indicated. C indicates positive control RNA; Y, yeast RNA. (C) HCD57 (lanes 1, 2), DA3-EPOR (lanes 3-6), or BAF3-EPOR (lanes 7-10) cells were cultured either continuously in EPO (c, lanes 3, 7) or in 10 ng/mL IL-3 overnight (lanes 4, 8), or deprived of EPO overnight and then stimulated with nothing (lanes 1, 5, 9) or EPO (lanes 2, 6, 10) for 4 hours. Y indicates yeast RNA (lane 11). (D) HCD57 cells were deprived of EPO for 18 hours and then pretreated with DMSO vehicle (lane 2), 5 and 50 μM PD98059 (lanes 4, 5), 1 or 10 μM U0126 (lanes 6, 7), 5 or 50 μM LY294002 (lanes 8, 9), or 2 and 20 μM SB203580 (lanes 10, 11) for 2 hours prior to addition of EPO for 4 hours (lanes 2-11). C indicates positive control RNA; Y, yeast RNA; P, undigested probe.