Figure 4

Induction of apoptosis in human astrocytomas by STAT3 RNAi. A. A172 cells were transfected with STAT3, GFP siRNAs, or mock transfected with oligofectamine (vehicle). After 72 hrs, cells were fixed in 4% paraformaldehyde and stained with 25 μg/ml Hoechst 33258 dye to visualize apoptotic nuclei. The percentage of apoptotic cells (small bright nucleus) was determined for each of the treatments by counting five visual fields and at least 100 cells. B. A172 cells were transfected with STAT3 siRNA or mock transfected with oligofectamine. Western blotting was performed 72 hrs post transfection. Blots were probed with α-STAT3, α-cleaved caspase 3 and α-β-actin antibodies. C. A172 cells were transfected with STAT3 siRNA (bottom panel) or mock transfected with oligofectamine (top panel). After 96 hours, cells were isolated and immunostained with EGFP coupled anti-annexin V antibody. Cells were sorted by FACS and annexin V positive cells counted. The region shaded in black (77%) corresponds the increase in annexin V positive cells relative to the mock transfected cells.