ion. DNA synthesis was plotted as percent of untreated control. In U266 cells there was a dose-dependent steady decrease reaching a decline of 70% DNA synthesis with 3 M at 24, 48, and 72 h. This effect was not observed in MM.1S cells where endogenous DNA proliferation was maintained at all concentrations and different time points. Effect of SGI-1776 treatment on autophagy in MM cell lines Autophagy, a catabolic process involved in cell survival, is characterized by acidic vesicular organelle formation26. Acridine orange staining is a dye that accumulates in acidic organelles. Measurement of this dye by flow cytometry is used to detect AVO formation. Cells were treated with the vehicle or SGI-1776 1 M and 3 M for 24, 48, and 72 h. U266 cells were treated with bafilomycin A1 for 30 min prior to acridine orange stating to block acidic vesicular formation and was used as a negative control. Similarly cells were treated PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844094 with rapamycin a known inducer of autophagy as a positive control. As expected, bafilomycin A1 inhibited the fusion of autophagosomes with lysosomes 
resulting in 3% – 2% acridine orange staining. Endogenous autophagy levels were less than 15% at all-time points, which remained after cells were treated with rapamycin. Interestingly, 1 and 3 M SGI-1776 treatment resulted in 47% and 53% acridine orange staining at 24 h. Similarly, a 28% and 59% staining was observed for 1 and 3 M SGI-1776 concentrations at 48 h; and 25% and 71% at 72 h. A parallel assay was done in MM.1S cell line and quantitated. As with U266, MM. 1S cell line also showed increased acridine orange stain in a dose- and time- dependent fashion, albeit at a lower level. Effect of SGI-1776 treatment on autophagy proteins Acridine orange staining demonstrated autophagy induction by SGI-1776 treatment. Hence, key proteins that monitor autophagy were evaluated by immunoblot analysis. U266 cells were treated as indicated and protein levels were measured for phosphorylation levels of AMPK at Thr172, LC3b. AMPK is a protein kinase sensitive to the levels of ATP in the cell. A decrease of the ATP cellular pool results in the phosphorylation of AMPK at Thr172 subsequently activating this kinase28. SGI-1776 treatment resulted in no visible change of phosphorylated AMPK at the amino acid residue Thr172. This suggests that SGI-1776 does not affect energy homeostasis and the order PR-619 autophagic pathway is being activated downstream of the ATP sensor AMPK. LC3b is an autophagic marker that is cleaved and lipidated to form LC3b-II which is bound to autophagosomes. Hence, LC3b conversion correlates with autophagosome formation and it is commonly used to monitor autophagy29. SGI-1776 treatment resulted in lipidation of LC3-I to LC3-II as shown by the visualization of a lower band around 17 KDa in a doseand time- dependent manner. Effect of SGI-1776 treatment on cell cycle in MM cell lines U266 and MM.1S cells were treated as indicated and analyzed for cell cycle profile. G1, S, and G2/M populations were quantitated in both cell lines. Similar experiments were performed three times and data were tabulated. There was no change in any population in either cell line at any concentration or time point, suggesting SGI-1776 treatment in MM cells does not affect the cell cycle profile. Effect of SGI-1776 treatment Pim kinase substrates NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Effect of SGI-1776 treatment on transcription proteins–U266 and MM.1S cells were treate