Figure 3

Ergosterol peroxide (EP) enhances expression of SHP-1 in U266 cells. (A) Cells were treated with 25 μM EP in the absence or presence of pervanadate (10, 25 or 50 μM) for 4 h. Whole cell extracts were prepared and subjected to Western blotting to determine level of phospho-STAT3 and STAT3. (B and C) Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h (B) and various concentrations of EP (0, 6.25, 12.5 or 25 μM) for 4 h (C). Whole cell extracts were prepared and subjected to Western blotting to determine SHP-1 expressoin. (D) Cells were treated with 25 μM EP for 0, 4, 8, 16 or 24 h. RT-PCR was performed to analyze mRNA expression of SHP-1. (E) Cells were treated with 25 μM EP for 4 h (left), or treated with 25 μM EP for 1 h followed by washing PBS to remove EP and resuspension in fresh medium (right). Western blotting was performed for SHP-1. (F) Cells were transiently transfected with either SHP-1 or scrambled siRNA (50 nM) for 48 h and then treated with 25 μM EP for 4 h. Western blotting was performed for phospho-STAT3 and STAT3.