PMC

Induction by Dox is shown for GFP in all cell lines, for (A)-GFP in the a1 clone, and for (B)-GFP in the b1 clone of NIH 3T3 cells by Western blotting as described in Methods. Note that some (A)-GFP and (B)-GFP are produced in a1 and b1 clones, respectively, in the absence of Dox.

p75ICD ((A)-GFP; a1 clone) potentiates induction of cell death (MTS assay) by H₂O₂ in NIH 3T3 cells. Deletion of the Chopper domain from p75ICD ((B)-GFP; b1 clone) restores the resistance of these cells to H₂O₂. Results shown are the mean ± SEM of three independent experiments performed.

Stable (B)-GFP transfectants were treated with vehicle or 6-OHDA in the absence or presence of Dox and studied by flow cytometry as for (A)-GFP transfectants shown in . The results of one experiment of two performed are shown. Control values are not siginificantly different (Student's t-test) between GFP(−) and GFP(+) cells. *P = 0.05 relative to GFP(−) cells.

Time course of activation of caspase-3 after treatment with 6-OHDA (150 μM, 1 hour exposure) in stable MCS, A37, and B2 clones. Activated caspase-3 levels were determined by Western blotting and optical densitometry of the resulting bands. Protection from caspase-3 activation is greatest in B2 cells and less robust in A37 cells.

(a) Light and fluorescent micrographs of stable HN33.11 cells expressing (A)-GFP or (B)-GFP, respectively. Results from two stable clones of each transfection pool are shown. Cells are treated with vehicle (Dox−) or 1 μg/mL Dox (Dox+). (b) Western blot of homogenates of stable clones showing endogenous production of full-length p75NTR (FLp75), Dox-induced, but “leaky” production of (A) and (B) and of the V5 tag. Control values for each clone do not differ significantly between GFP(−) and GFP(+) cells (Student's t-test). ***P < 0.001 relative to GFP(−) cells.

Hoechst dye staining of MCS, A37, and B2 cells 48 hours after treatment with 6-OHDA (150 μM, 1 hour exposure). Two-hundred total cells were counted in each treated well to determine the percent of the cells that exhibited nuclear condensation and/or fragmentation and margination of DNA. P values for A37 and B2 cells are calculated by Student's t-test relative to MCS cells. Protection from morphologic apoptosis is greatest in B2 cells and less robust in A37 cells.

(a) Concentration-survival curve of HN33.11 cells to 6-hydroxydopamine (6-OHDA). Cell survival was determined by MTS assay as described in Methods. (b) Effects of Dox-induced expression of (A)-GFP on induction cell death by 6-OHDA in HN33.11 cells (clones A37 and A38). The stable lines indicated were plated at 5×10⁵ cells per 60 mm dish and exposed to 6-OHDA (300 μM) for 1 hour and incubated for 48 hours prior to analysis by flow cytometric quantitation of sub-G1 nuclei. The results of one experiment of three performed are shown.

(a) Results of transfection of HN33.11 cells with green fluorescent protein- (GFP-) linked empty plasmid (C2eGFP) or GFP-linked multiple cloning site in the same plasmid with a doxycycline- (Dox-) inducible internal ribosome entry site (MCSiresGFP). Dox elicits production of GFP in the MCSiresGFP-transfected cells, but not in C2eGFP-transfected cells. (b) Dox-inducibility of MCSiresGFP-linked expression constructs for (A) and (B) as defined in . Open bars: cells maintained in the absence of Dox; closed bars: cells maintained in the presence of Dox (1 μg/mL). Results shown are the mean ± SEM of three independent experiments performed. ***P < 0.001; **P < 0.01 relative to absence of Dox, Student's t-test.

Regulating p75ICD expression using the pBIG2i Tet-inducible expression system. Full-length p75ICD (ICD-A) or the N-terminal deletion mutant (ICD-B) was expressed from the bidirectional tetracycline regulated vector pBIG2i. In the presence of ligand (doxycycline), the reverse transactivator (rtTA) expressed from the thymidine kinase promoter (TK) binds to the heptemerized tetO sequence activating transgene expression from both the minimal TK and CMV promoters. Inclusion of the IRES-eGFP cassette facilitates the analysis of p75ICD expressing clones. Domains included in the full-length ICD include the amino terminal “chopper domain”, tandem PEST sequence, Traf2- and PDZ-binding domains, and the carboxyl terminal death domain. In addition to using IRES-mediated GFP expression, both ICD expression constructs contain the V5 epitope to monitor inducible ICD transgene expression.