Fig. 7. Decreased survival of transgenic mice after transverse aortic constriction (TAC). Survival rates in the days following TAC or sham operation (sham) in 3×-DN–14-3-3 mice (TG) and their non-transgenic littermates (NTG).

Fig. 8. Increased cardiomyocyte apoptosis in transgenic mice after TAC. Hearts were isolated from 3×-DN–14-3-3 transgenic mice and their non-transgenic littermates 5 days after TAC, fixed in paraformaldehyde, embedded in paraffin, sectioned with a microtome and analyzed by TUNEL assay. Sections of ventricular tissue obtained from (A) a sham-operated non-transgenic mouse, (B) a non-transgenic mouse obtained 5 days after TAC, (C) a 3×-DN–14-3-3 transgenic mouse obtained 5 days after sham operation and (D) a 3×-DN–14-3-3 transgenic mouse obtained 5 days after TAC. TUNEL-positive nuclei are indicated by arrows.

Fig. 5. Targeted expression of DN–14-3-3η to postnatal murine cardiac tissue. Detection of DN–14-3-3η protein in transgenic mouse cardiac tissue. Immunoblotting was performed on ventricular extracts of two 3×-DN–14-3-3 mice (TG1 and TG2) and a non-transgenic (NTG) littermate. Equal amounts of total protein were loaded in each lane. Murine monoclonal anti-Myc-1-epitope antibody, rabbit polyclonal anti-pan-14-3-3 antibody and anti-ERK MAPK primary antibodies were used. Similar results were obtained in three hearts in each group.

Fig. 1. Protein levels of mutant forms of 14-3-3 in NIH 3T3 cells. (A) Immunoblot analysis of protein lysates. NIH 3T3 cells were stably transfected with HA epitope-tagged versions of DN–14-3-3ζ, NES–14-3-3ζ and WT–14-3-3ζ, or with vector alone. Protein lysates from stably transfected NIH 3T3 cells (DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ, Vector) were separated by SDS–PAGE and analyzed by immunoblotting with an anti-HA epitope primary antibody. (B) DN–14-3-3η binds to native WT–14-3-3ζ. NIH 3T3 cells were stably transfected with Myc-1 epitope-tagged DN–14-3-3η or with vector alone. Cytosolic extracts of transfected cells were used to generate anti-Myc-1 immunoprecipitates that were analyzed by immunoblotting with a highly specific anti-14-3-3ζ antibody. The anti-14-3-3ζ antibody does not recognize DN–14-3-3η (S.Zhang et al., 1999).

Fig. 6. Increased basal JNK1 and p38 MAPK activity in DN–14-3-3 cardiac tissue. (A) JNK1 activity was assessed in the ventricular tissue of three 3×-DN–14-3-3 mice and three non-transgenic littermates. Immunoblots of cytosolic extracts were analyzed by the use of an anti-phospho JNK antibody. Equal amounts of total protein were loaded in each lane. (B) p38 MAPK activity was assessed in the ventricular tissue of three 3×-DN–14-3-3 mice and three non-transgenic littermates. Immunoblots of cytosolic extracts were analyzed by the use of an anti-phospho p38 MAPK antibody. Equal amounts of total protein were loaded in each lane. Densitometric analysis of immunoreactive bands in (A) and (B) was performed by the use of NIH Image software and data are expressed as the mean signal intensity ± SEM.

Fig. 3. Increased sensitivity to UVC-, serum deprivation- and TNF-α-induced apoptosis in DN–14-3-3ζ-transfected cells. (A and B) Killing curves of transfected (Vector, DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ) NIH 3T3 cells (A) 48 h after UVC irradiation and (B) after 48 h of serum deprivation. Cells were analyzed by the Trypan blue exclusion method. At least 100 cells were analyzed for each condition. Results are the mean ± SEM of three experiments. (C) DNA fragmentation assays before (–) and after UVC irradiation (+). Untransfected (3T3) or stably transfected (DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ) NIH 3T3 cells were irradiated with 180 J/m² UVC, incubated for 48 h, and then genomic DNA was extracted from cells to assess DNA fragmentation by agarose gel electrophoresis. These results are representative of four separate experiments. (D and E) Killing curves of transfected (Vector, DN–14-3-3ζ, DN–14-3-3η, NES–14-3-3ζ, WT–14-3-3ζ) NIH 3T3 cells after 12 h of stimulation with (D) 100 ng/ml TNF-α and (E) 50–100 μM hydrogen peroxide or 100 μM etoposide. Cells were analyzed by the Trypan blue exclusion method. At least 100 cells were analyzed for each condition.

Fig. 4. Treatment with SB202190 or transfection with dominant-negative p38α inhibits UVC irradiation-induced apoptosis in DN–14-3-3ζ-, DN–14-3-3η- and NES–14-3-3ζ-transfected NIH 3T3 cells. NIH 3T3 cells were stably transfected with DN–14-3-3ζ, DN–14-3-3η or NES–14-3-3ζ, and some were treated with SB202190 (20 μM) (+) or with vehicle (–) for 1 h prior to irradiation with 180 J/m² UVC and then incubated for 48 h. (A) Killing curves of transfected NIH 3T3 cells 48 h after UVC irradiation. Cells were analyzed by the Trypan blue exclusion method. At least 100 cells were analyzed for each condition. Results are the mean ± SEM of three experiments. (B) DNA fragmentation assays performed 48 h after UVC irradiation in transfected NIH 3T3 cells that were treated with SB202190 (+) or with vehicle (–). These results are representative of three separate experiments. (C) Killing curves of dominant-negative p38α-transfected NIH 3T3 cells 48 h after UVC irradiation. At 36 h prior to UVC irradiation, NIH 3T3 cells that were stably transfected with DN–14-3-3ζ or DN–14-3-3η were transiently transfected with 2 μg of vector or with dominant-negative human p38α. Doubly transfected cells were analyzed for total p38 protein levels and p38 activity by immunoblotting with anti-p38 and anti-phospho-p38 antibodies. Cells were analyzed by the Trypan blue exclusion method. At least 100 cells were analyzed for each condition. Results are the mean ± SEM of three experiments.

Fig. 2. Mutant forms of 14-3-3 differentially affect MAPK signaling pathways. (A) Transfection of NIH 3T3 cells with DN–14-3-3ζ but not with NES–14-3-3ζ or WT–14-3-3ζ blocks ERK MAP kinase activation. Cells were deprived of serum for 24 h (–) and some (+) were treated with 10% FCS for 10 min. Protein lysates from stably transfected cells (DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ) were separated by SDS–PAGE and analyzed by immunoblotting with a highly specific, anti-phospho-ERK antibody (upper panel). Equal amounts of total protein were loaded in each lane. Lysates were also analyzed in parallel by immunoblotting with an anti-ERK (lower panel) antibody to control for protein content. These immunoblots are representative of five separate experiments. (B) DN–14-3-3ζ, DN–14-3-3η and NES–14-3-3ζ promote Ask1 activation. NIH 3T3 cells stably transfected with DN–14-3-3ζ, DN–14-3-3η, NES–14-3-3ζ or WT–14-3-3ζ were grown in the presence of serum. Anti-Ask1 immunoprecipitates derived from stably transfected NIH 3T3 cells were assayed for kinase activity by the use of MBP as a substrate in the presence of [γ-³²P]ATP. Equal amounts of total protein were used for each immunoprecipitate. Proteins were separated by SDS–PAGE and radiolabeled MBP was detected by autoradiography. Bands were quantitated by densitometry using NIH Image software, and each column reflects the mean signal intensity ± SEM of three determinations. (C) DN–14-3-3ζ and NES–14-3-3ζ promote JNK1 activation. Cells were deprived of serum for 24 h and some were treated with 10% FCS for 10 min. Protein lysates from stably transfected cells (DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ, Vector) were separated by SDS–PAGE and analyzed by immunoblotting with a highly specific, anti-phospho-JNK antibody (upper panel). Equal amounts of total protein were loaded in each lane. Lysates were also analyzed in parallel by immunoblotting with an anti-JNK (lower panel) antibody to control for protein content. These immunoblots are representative of three separate experiments. (D) DN–14-3-3ζ and NES–14-3-3ζ promote p38 MAPK activation. Cells were deprived of serum for 24 h and some were treated with 10% FCS for 10 min. Protein lysates from untransfected (3T3) or stably transfected cells (DN–14-3-3ζ, NES–14-3-3ζ, WT–14-3-3ζ) were separated by SDS–PAGE and analyzed by immunoblotting with a highly specific, anti-phospho-p38 (upper panel) antibody. Equal amounts of total protein were loaded in each lane. Lysates were also analyzed in parallel by immunoblotting with an anti-p38 MAPK (lower panel) antibody to control for protein content. These immunoblots are representative of three separate experiments.