Over-expression of IER5 inhibited AML cell proliferation.

A. The cell proliferation of U937 cells, transfected with IER5 cDNA or treated with TMPP, was measured by counting cells using a hemocytometer (upper panel). Cell counting was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. *P<0.01 compared with untransfected control cells. Cell viability of the IER5 over-expressing U937 cells was assessed by counting of viable cells using trypan blue staining at 72 h, starting 3 days after DNA transfection (bottom panel). B. QRT-PCR analysis of IER5 mRNA expression in untreated cells, IER5 cDNA-transfected cells, and TMPP-treated U937 cells. QRT-PCR was started 3 days after transfection, and was performed every 24 h for 3 days. Data are shown as means ± S.D. of triplicate cultures and are representative of three independent experiments. The levels of the quantified RT-PCR products were normalized to GAPDH expression in the same sample and were then expressed relative to the mRNA level of a normal control, which was assigned a value of 1. *P<0.01 compared with untransfected control cells. The protein expression of IER5 in cells was analyzed after 3 days of culture (bottom panels). Blotting of Actin was used as a loading control. C. The cell cycle distribution of U937 cells that were transfected with control DNA, IER5 cDNA, scrambled shRNA, IER5 shRNA #1, IER5 shRNA #1, or were treated with TMPP, was analyzed using flow cytometric analysis. The transfected or TMPP-treated U937 cells were harvested after 3 days. The fraction of cells in the G1, S and G2/M stage of the cell cycle was determined. Data are shown as means ± S.D. of triplicate cultures. The IER5 mRNA expression of the cells is shown at bottom and was assessed using RT-PCR. The RT-PCR results are representative of three independent experiments. Data are shown as means ± S.D. of triplicate cultures. *P<0.01 compared with control cells. D and E. Cell cycle analysis (D) and changes of mitochondrial membrane potential (ΔΨm) (E) in IER5 overexpressed or TMPP treated AML cells. U937 cells were transfected with IER5 cDNA or treated with TMPP (5 µM). Mitochondrial membrane potential was determined 3 days after transfection or TMPP treatment by staining of the cells with DiOC6 followed by flow cytometric analysis. The FACS results are representative of three independent experiments. NC; Negative control.