Figure 4.

Effect of TS depletion on apoptosis in lung cancer cells. (A) The indicated cell lines were transfected with nonspecific (NS) or TS-1 siRNAs for 48 or 72 h and were then fixed, stained with propidium iodide, and subjected to flow cytometry for quantitation of the sub-G₁ population. Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results. (B) Cells were transfected with NS or TS-1 siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to TS, PARP, and β-actin. The positions of intact and cleaved forms of PARP are indicated. (C) Cells were transfected with NS or TS-1 siRNAs for 48 or 72 h, lysed, and assayed for caspase-3 activity. Data are expressed relative to the value for cells transfected with NS siRNA and are means±s.d. from three independent experiments. (D) Cells were incubated for 2 h with or without ZVAD-FMK (50 μM), transfected with NS or TS-1 siRNAs for 48 or 72 h (in the continued absence or presence of ZVAD-FMK), and then evaluated for the size of the sub-G₁ population as in (A). Data are means±s.d. of triplicates from experiments that were repeated on two additional occasions with similar results. ^*P<0.05 for the indicated comparisons. Transfection with the NS siRNA had no substantial effects on these markers of apoptosis compared with untreated cells.