JunD Protects Cells from p53-Dependent Senescence and Apoptosis Jonathan B. Weitzman, Laurence Fiette, Koichi Matsuo, Moshe Yaniv  Molecular Cell  Volume 6, Issue 5, Pages 1109-1119 (November 2000) DOI: 10.1016/S1097-2765(00)00109-X

Figure 1 Growth Defect of Primary Fibroblasts Lacking JunD (A) Fibroblasts from individual wild-type (d+/+) or mutant (d−/−) embryos were placed in culture (passage 2) at equal cell densities and passaged every 3 days at the same density. Experiments were performed on multiple individually isolated embryo-derived fibroblast cultures and duplicate plates were counted. (B) Early passage primary MEFs were plated at low density (10,000 cells per 10 cm plate) and incubated for 2 weeks with regular changes of media. Cell colonies (>30 cells) were then fixed, stained, and counted. Experiments were performed in triplicate for three individually generated MEF cultures. (C) Fibroblasts at passage 6 were plated and then incubated in different serum concentrations for 36 hr, after which cells were counted. The changes in cell number are shown for duplicate samples of wild-type, heterozygote, or homozygote mutant cells. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 2 Senescence Features of JunD−/− Fibroblasts (A) Staining for β-galactosidase activity of early passage MEFs. Staining was performed at pH 6.0 to detect acidic SA-β-gal activity and at pH 7.4 to detect nuclear-restricted, bacterial NLS-LacZ activity. For clarity, phase contrast (on left) and bright light (right) images are shown for each genotype. (B) Upregulation of p53 and p19Arf in JunD−/− cells. Immunofluorescence staining of early passage MEFs. The enlarged cells in JunD−/− cultures showed an increase in nuclear p53 and nucleolar p19Arf staining (arrows), which was not observed in wild-type fibroblast cultures. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 3 The Role of Ras and p53 in the Growth Defect of JunD−/− Cells (A) Mutant JunD−/− or wild-type MEFs were transduced with H-ras V12-expressing pBabe viruses in the presence or absence of viruses encoding JunD. The percentage of SA-gal-positive (pH 6.0) cells was determined 48 hr after selection and replating. The results represent mean ± SD of quadruplicate measurements. (B) Growth of MEFs generated from crosses between JunD+/−p53+/− mice. The four relevant genotypes were examined: wild-type cells (d+/+p+/+), single mutant cells (d+/+p−/− or d−/−p+/+), and double mutant cells (d−/−p−/−). Cells of each genotype were plated at very low density (3000 cells per 10 cm plate), and the colony (> 30 cells) forming potential was measured 2 weeks later. The results represent the mean ± SD of triplicate plates for three individual cultures for each genotype. (C) Growth curves of MEFs with four different p53 and JunD combined genotypes. The removal of functional p53 overcomes the growth defect observed upon junD disruption. Results represent the mean of duplicate values ± SD. (D) Immunofluorescence staining of early passage MEFs on a p53-null background. There is an increase in nucleolar p19Arf staining in p53−/− cells, which was not observed in wild-type fibroblast cultures. Furthermore, nearly all JunD−/−p53−/− double mutant cells express high p19Arf levels. (E) Staining for β-galactosidase activity under different pH conditions. JunD−/− cells on a p53-null background (p−/−d−/−) lose the specific acidic SA-β-gal staining (pH 6.0), but maintain nuclear staining at pH 7.4 due to the inserted bacterial LacZ gene. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 4 Growth Characteristics of Immortalized Cell Lines (A) Growth of 3T3-derived immortalized cells lines. Cells derived from JunD−/− embryos grow faster than heterozygotes of wild-type lines. All points represent the mean of duplicate cultures. Cells were plated at 100,000 cells per plate and counted at 3-days intervals. (B) Saturation density. Immortalized cell lines (two for each genotype) were cultured in different serum concentrations and allowed to grow for 1 week until they reached confluence. Cells were then trypsinized and cell numbers counted. (C) Western blot analysis of whole extracts from two immortalized cell lines from wild-type (d+/+) or mutant (d−/−) cultures. Analysis was performed using antibodies specific for the JunD protein, the cell cycle regulator cyclinD1, and β-tubulin as a control. (D) Photographs of confluent monolayers of wild-type (+/+) or mutant (−/−) 3T3 cell lines. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 5 The UV Hypersensitivity of JunD−/− Primary Fibroblasts Is p53-Dependent (A) Early passage primary MEFs were exposed to different UV irradiation and the extent of apoptotic nuclei determined (16 hr postirradiation). The values represent the mean of five random fields. Similar results were obtained when viability of surviving cells was determined by trypan blue dye exclusion. (B) Cells were irradiated with 10 J/m2 UV, and the extent of apoptotic nuclei was determined 16 hr later. Removal of the p53 gene overcame the UV hypersensitivity observed in JunD−/− cells. Values represent the mean ± SD for five random fields. Two independent cell cultures are shown for each genotype. (C) Clonogenicity assays showing two cultures of p−/−d−/− MEFs compared a p−/−d+/+ single mutant culture, treated with increasing amounts of UV irradiation. Survival of colonies of the two genotypes was indistinguishable. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 6 TNFα-Induced Cytotoxicity in Primary Fibroblasts Lacking JunD (A) Two independent cultures of primary fibroblasts from wild-type mutant embryos were treated with TNF-α (10 ng/ml). Cell numbers were calculated at different times after treatment (n = 5). Similar results were obtained when cell viability was investigated using exclusion of the dye trypan blue. (B) Cell death of TNF-treated cultures was assessed by examining nuclear fragmentation revealed by staining with Hoechst dye. Results are shown for two independent cell cultures for each genotype following 48 hr of treatment. Results represent mean ± SD of five different fields. (C) Micrographs of primary MEFs treated with TNF-α (10 ng/ml). Phase contrast and Hoechst staining images are shown for each genotype. Arrows indicate cells displaying apoptotic morphology and condensed nuclei. (D) Dose response to TNF-α-induced cell death measured at 16 hr posttreatment with cytokine. (E) JunD−/− or wild-type MEFs were transduced with pBabe viruses encoding H-ras V12 (+R), alone or in combination with JunD-pBabe (+R+D). After selection, cultures were treated with murine TNF-α (10 ng/ml), and apoptosis was determined after 20 hr of incubation. Results represent mean ± SD of four independent fields. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)

Figure 7 Increased LPS-Induced Hepatitis in Mice Lacking JunD (A) Survival curve for JunD−/− (triangles) or wild-type animals (squares) following intraperitoneal co-injection of sublethal doses of LPS (0.5 μg/kg) with GalN (900 mg/kg). (B and C) Histological analysis of livers isolated from LPS-treated wild-type (B) and JunD−/− animals (C). Paraffin sections (4μm) were stained with hematoxylin-eosin. Magnification 400×. Apoptotic hepatocytes (C) appear as eosinophilic ovoid masses. In most of the nuclei, the chromatin is condensed, deeply basophilic, and fragmented, and appears as peripheral crescents, spots, or pycnotic bodies. Molecular Cell 2000 6, 1109-1119DOI: (10.1016/S1097-2765(00)00109-X)