sembly compared to lenalidomide alone, suggesting that rhEPO and lenalidomide augment membrane raft assembly by distinct mechanisms. To confirm that the increase in raft fractions after lenalidomide exposure reflected induction of raft formation, we assessed the number and size of lipid rafts by GM1 immunofluorescence. Immunofluorescence data confirmed the induction of membrane rafts by lenalidomide. Collectively, these results demonstrate that LEN is able to stimulate raft PBTZ 169 manufacturer formation and aggregation independent of cytokine or receptor stimulation. To determine if lenalidomide also induces rafts in primary erythroid progenitors, BM-MNC from MDS patients and normal donors were treated with 1 uM lenalidomide for 1 h prior to raft and surface marker staining. Analysis of CD71+/cKit+ erythroid cells from MDS patients demonstrated an increase in the total raft number per cell that did not reach statistical significance. However, the size of the membrane raft aggregates significantly increased after LEN treatment . These results indicate that lenalidomide induces aggregation of smaller raft clusters into larger membrane signaling platforms that may potentiate EpoR signaling. In normal erythroid cells, lenalidomide did not significantly alter the 4 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors size of raft aggregates, suggesting that the potentiating effect on raft assembly in MDS arises from actions on aberrant mechanisms limiting raft formation in raft deficient MDS erythroid cells. Of the 11 MDS patients analyzed, 8 received treatment with lenalidomide, including 5 responders and 3 nonresponders. Membrane raft size was significantly larger in the erythroid progenitors of responding patients compared to non-responders . Furthermore, lenalidomide increased raft size in responders and nonresponders with greater raft induction in 
responding patient progenitors, . Lenalidomide recruits EpoR signal effectors into raft fractions Our prior studies of rhEpo showed that in addition to inducing raft formation and aggregation, rhEpo induced the recruitment of EpoR and its signaling intermediates JAK2, STAT5, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683642 and Lyn kinase into raft fractions. Treatment with rhEpo also partitioned the negative regulator and transmembrane protein, 6 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors tyrosine phosphatase, CD45, out of the raft fractions, thereby potentiating fidelity of the EpoR signal. To determine whether lenalidomide treatment effected raft constituents, we treated UT7 cells with lenalidomide and isolated both the raft and non-raft fractions after ultracentrifugation. These fractions were then probed by western blot analysis. Lenalidomide induced the recruitment of EpoR into lipid raft fractions after 1 hr of drug exposure. Furthermore, JAK2, STAT5, and Lyn kinase showed increased partitioning with GM-1 after lenalidomide treatment, indicating recruitment of signaling effectors into discrete signaling platforms. Lastly, we found that treatment with lenalidomide also redistributed CD45 to non-raft fractions, which may further enhance signal fidelity. Lenalidomide enhances EpoR signaling in erythroid progenitors To determine if lenalidomide enhances EpoR signaling, we treated UT7 cells with rhEpo PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682429 alone and after treatment with 1 mM lenalidomide. JAK2 phosphorylation following rhEpo stimulation was enhanced and prolonged following lenalidomide pretreatment. Similar findings were observed with STAT5 phosphorylation in whi
