V-ANedd rescues PC12 cells from serum deprivation, but not from SOD1 downregulation. A, V-ANedd differentially protects PC12 cells from serum deprivation. For serum deprivation (4 left-hand bars) cells were washed extensively, as described in Materials and Methods, and the indicated additives (100 ng/ml NGF, 400 nmV-ANedd, or 200 nmV-ICE_inh) were added at the time of plating in serum-free RPMI 1640 medium. For SOD1 downregulation (4 right-hand bars), PC12 cells were replated on fresh collagen-coated 24-well dishes in complete medium (RPMI 1640 medium with 10% horse serum/5% fetal bovine serum) with 50 nmV-ASOD1 (vector-linked antisense oligonucleotide to SOD1). Additives (800 nmV-ANedd and 25 nmV-ICE_inh) were included as indicated. Control cells were in complete medium. Cultures were incubated for 24 hr and lysed, and the number of intact nuclei was counted. The numbers of surviving cells are expressed relative to the number in the control cultures (designated as 100). Here, as in past studies (Greene and Tischler, 1976; Rukenstein et al., 1991; Troy et al., 1996a,b), NGF or complete medium promotes survival of all cells initially plated. Experiments were performed in triplicate wells, and data are expressed as mean ± SEM. B, Dose–response curve for protection from serum deprivation by V-ANedd. PC12 cells were washed for trophic factor deprivation and plated in serum-free medium with the indicated concentrations of V-ANedd. Cell survival relative to the number present with the addition of NGF was measured at 1 d. C, V-ANedd does not block the V-ASOD1-induced increase of IL-1β production. PC12 cells were plated with the indicated additives (50 nmV-ASOD1, 25 nmV-ICE_inh, and 800 nmV-ANedd). Controls contained complete medium. After 20 hr, media were removed, and IL-1β was measured by ELISA with the Intertest-1βX kit. Data are expressed as mean ± SEM (n = 3).