FIGURE 4.

TNF-induced apoptotic cascade is impaired in CLN2-deficient cells. A, expression of cell surface TNFR1, FADD, and procaspase-8 in control and LINCL fibroblasts, as assessed by flow cytometry and Western blot, respectively. Fluorescence intensity of individual cells is reported as means of arbitrary fluorescence units (mean fluorescence intensity (MFI)) ± S.E. of three independent experiments. Control and LINCL-transformed fibroblasts (B) for the indicated times or Epstein-Barr virus-transformed lymphoid cell lines (C) were incubated in medium containing 1% FCS with 5 ng/ml TNF and 50 μg/ml cycloheximide or 50 ng/ml anti-CD95, respectively. Cells were then harvested, and DEVDase activity was determined. Data are means ± S.E. of three independent experiments. D, control and LINCL fibroblasts were treated for the indicated periods with 50 μg/ml cycloheximide and 5 ng/ml TNF. Cell lysates were prepared and analyzed by Western blotting for Bid proform disappearance, cytochrome c release, caspase-8, -9, and -3 cleavage, Hsp70, and cIAP1 expression. An anti-β-actin was used as a control for protein loading. Results are representative of three independent experiments.