TBRI inhibition can inhibit TGF-β–mediated cell-cycle arrest of CD34⁺ cells. Equal numbers of BM CD34⁺ cells were grown in the presence of SCF, TPO, and FLT-3L and were pretreated with DMSO or 0.5 μM SD-208 for 1 hour before TGF-β1 (0.5 ng/mL final concentration) was added. On days 3, 5, and 7, cell aliquots were taken and viable cell concentration was determined using Guava ViaCount. The experiment was repeated at least 3 times (using multiple donors of CD34⁺ cells) and means plus or minus SEM is shown (A). CD34⁺ cells were treated with DMSO or SD-208 in the presence or absence of TGF-β1 for 7 days. Cell-cycle distribution of CD34⁺ cells (gated with PE-conjugated CD34 antibody) was determined on day 7 using the APC BrdU Flow Kit and the LSR-II flow cytometer (BD Biosciences; B; representative sample, C). Error bars represent SEM. CD34⁺ cells were treated with DMSO or SD-208 in the presence or absence of TGF-β1 as described above. After 24 hours, cDNA was prepared and hybridized on a cDNA microarray. Selected cell-cycle progression genes that were down-regulated by TGF-β by 2-fold were validated at the protein level by Western blotting. CD34⁺ cells were treated with DMSO or SD-208 in the presence or absence of TGF-β1 as described above. Cells were collected at the indicated time points and lysed in radioimmunoprecipitation assay (RIPA) buffer. Equal protein was separated on a 10% Bis-Tris sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membrane and immunoblotted with the antibodies (D). GAPDH levels were used as protein loading controls, and p-smad2 was used as positive control for TGF-β1 stimulation.