Figure 2

Induction of apoptosis and inhibition of phosphorylation of RNA Pol II and phosphor-mTOR by SNS-032. (A) HL-60, KG-1 and HEL cells were treated with SNS-032 for 24 h, and apoptosis was assessed by flow cytometry after staining of the cells with annexin V-FITC and propidium iodide. (B) Three AML cell lines were treated with SNS-032 at the indicated doses for 6 h. PBS was used as a negative control. The total cell lysates (50 μg of protein per lane) were analyzed by Western blotting with antibodies against RNA pol II, RNA pol II carboxy terminal domain phosphor-serine 2 and phosphor-serine 5, mTOR, phosphorylated form of mTOR on Ser2448 and Ser2481. Anti-β-actin was used as a loading control. (C) HL-60 cells were treated SNS-032 at the indicated doses for 6 h. The endogenous levels of mTOR protein phosphorylated at Ser2448 and were detected using solid phase sandwich enzyme-linked immunosorbent assay. (D) HL-60 cells were treated with SNS-032 (50–400 nM for 6 h). whole-cell lysates were subjected to Western blotting to assess phosphorylation and protein expression of NDRG1, p38MAPK, STAT3/5, and ERK1/2.