p38 Inhibitor SCIO-469 can reverse TNF-mediated myelosuppression. MDS1 cells were pretreated for 1 hour with vehicle (–) or 1.0 μM SCIO-469 (+) and then induced with either 1 ng/mL TNFα or 5 ng/mL TGFβ for 30 minutes. The p-p38 and total p38 levels were analyzed by Western blotting. Bar graph represents p-p38 levels relative to total p38 in each sample (A). Immunomagnetically selected bone marrow–derived CD34⁺ cells were differentiated into hematopoietic progenitors at the CFU-E stage of maturation as described before.³⁰  These cells were treated with 20 ng/mL TNFα or 10 000U/mL IFN-γ in the presence and absence of 100 nM SCIO-469. Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with an antibody against the phosphorylated form of MapKapK-2 (threonine 334). The same blot was stripped and reprobed with an antibody against total MapKapK-2, to control for protein loading (B). Primary bone marrow–derived CD34⁺ cells were grown in cytokine-enriched liquid media in the presence and absence of 20 ng/mL TNFα and SCIO-469 (100 nM) for 24 hours. The percentages of apoptotic and dead cells were determined by staining with mixture of Annexin V–Alexa Fluor 488 and nucleic acid dye, Sytox green, respectively (Vybrant Apoptosis Kit; Molecular Probes) (C). Mean of 3 independent experiments showed significant decrease in TNFα-mediated apoptosis in the presence of SCIO-469 (P = .01, paired t test). BM CD34⁺ progenitors were cultured with TPO, Flt3L, and SCF with or without 20 ng/mL TNFα and in the presence and absence of 500 nM SCIO-469 for 6 days. BrDU incorporation was evaluated against the amount of 7-AAD by flow cytometry to determine the percentage of subpopulation at each cell-cycle stage in a gated population of CD34⁺ cells. Results from 3 experiments were used to compare the proportion of cells in G₀/G₁ and S phase of cell cycle by using 2-tailed t test. (D) Primary bone marrow–derived CD34⁺ cells were cultured in methylcellulose in the presence and absence of 20 ng/mL TNFα and SCIO-469. Colonies were scored on day 14. Results are expressed as mean ± SEM of 3 independent experiments (E). Treatment with SCIO-469 led to a significant reversal of TNF-mediated myelosuppression (P = .04, t test).