Effect of overexpression of gelsolin on timing of apoptosis of growth factor-dependent cells induced by cytokine deprivation.

Overexpression of gelsolin did not delay apoptosis of growth factor-dependent cells induced by cytokine deprivation, or prevent its enhancement by jasplakinolide, despite appropriate cleavage. (A) Whole cell lysates (1.25 × 10⁶ cell equivalents) of transfected CTLL-20 cells were separated by SDS-PAGE and immunoblotted for gelsolin and for actin. Serum was used as a positive control. (B) Transfected CTLL-20 cells were incubated for 15 hours in the absence of IL-2 and in the presence of 100 nmol/L jasplakinolide (closed bars) or its vehicle, 0.02% Me₂SO (open bars). After incubation, cells were fixed and stained with propidium iodide for quantification of apoptosis by nuclear morphology. Results are representative of 2 independent experiments. (C, D) Whole cell lysates (2 × 10⁶ cell equivalents) of transfected Ba/F3 cells deprived of IL-3 for the indicated periods of time were separated by SDS-PAGE and immunoblotted for human (C) or murine (D) gelsolin. The time-dependent appearance of the 46 kd gelsolin cleavage product is shown. In (D) there was a slight loss of the cell sample at 8 hours. Results are representative of 2 independent experiments. (E) Parallel samples of transfected Ba/F3 cells deprived of IL-3 for the indicated periods of time were fixed and stained with propidium iodide for quantification of apoptosis by nuclear morphology. Closed circles represent Ba/F3 cells that overexpress gelsolin, while open circles represent the vector control. SDs were calculated as described in “Materials and methods.” The slight difference between the 2 cell lines at 8 and 12 hours was not evident by trypan blue exclusion.