Immunohistochemical staining of activated caspase-3 in skin samples. Skin samples were taken from mice of the following genotypes: nontransgenic, ARF^+/+ (A), nontransgenic, ARF^−/− (B), line E2F1.0, ARF^+/+ (C), and line E2F1.0, ARF^−/− (D). Sections were immunohistochemically stained for the activated form of caspase-3 and photomicrographed.

Morphological changes in apoptotic MEFs infected with AdE2F1. Early-passage MEF cultures isolated from ARF^+/+ (A and C) and ARF^−/− (B and D) sibling embryos were infected with AdGFP (A and B) or AdE2F1 (C and D) at an MOI of 100. At 24 h postinfection, the cells were stained with DAPI and examined microscopically. The nuclei of apoptotic cells appear condensed, abnormally shaped, and/or fragmented.

Inactivation of ARF enhances apoptosis in K5 E2F1 transgenic epidermis. (A) The TUNEL assay was performed on skin sections from nontransgenic (NonTg) and K5 E2F1 transgenic (line E2F1.0) mice that were either wild type for ARF (ARF^+/+) or null for ARF (ARF^−/−). The average number of TUNEL-positive cells per 10 mm of skin is presented for samples taken from at least four independent mice in each group. (B) Skin sections from the same mice as above were immunohistochemically stained for the activated form of caspase-3. The average number of caspase-3-positive cells per 10 mm of linear skin is presented. (C) Caspase-3 immunohistochemistry was performed on skin section from nontransgenic (NonTg) and K5 E2F1 transgenic (line E2F1.1) mice that were either wild type for ARF (ARF^+/+) or null for ARF (ARF^−/−). The average number of caspase-3-positive cells per 10 mm of linear skin is presented from samples taken from at least four independent mice in each group. Bars represent standard error.

Overexpression of E2F1 and inactivation of ARF cooperate to stimulate proliferation in mouse epidermis. Line E2F1.1 transgenic mice (E2F1.1) and nontransgenic (NonTg) sibling controls that were either wild type for ARF (ARF^+/+) or null for ARF (ARF^−/−) were injected with BrdU and allowed to incorporate the BrdU for 20 min before being sacrificed. Skin sections were immunohistochemically stained for BrdU, and the average percentage of cells in the interfollicular epidermis positive for BrdU from at least four independent mice in each group was calculated. The E2F1.1, ARF^−/− group was significantly different from the other groups by one-way analysis of variance with a Tukey Honestly Significantly Different Post-Hoc test with a significance level of 0.05.

Inactivation of ARF enhances apoptosis and proliferation induced by E2F1 in MEF cultures. (A) Early-passage primary MEF cultures wild type or null for ARF were infected with AdGFP or AdE2F1 at an MOI of 100. At 24 h postinfection, the cells were ethanol fixed and stained with DAPI. Nuclei that appeared condensed, fragmented, or abnormally shaped were scored as apoptotic. The average percentage of cells that were apoptotic from four independent experiments is presented. (B) Infection of MEF cultures was performed as for panel A except that an MOI of 50 was used. The cells were fixed 24 h postinfection following a 1-hour incubation in 10 mM BrdU. The cells were immunohistochemically stained using an antibody specific for BrdU and microscopically examined. The average percentage of cells incorporating BrdU in three separate experiments is presented.

Inactivation of ARF reduces apoptosis in K5 Myc transgenic epidermis. (A) The TUNEL assay was performed on skin sections from young adult nontransgenic (NonTg) and K5 Myc transgenic mice (line MM5) that were either wild type for ARF (ARF^+/+) or null for ARF (ARF^−/−). The average number of TUNEL-positive cells per 10 mm of skin is presented for samples taken from at least four independent mice in each group. (B) K5 Myc transgenic and nontransgenic (NonTg) control mice, wild type for ARF (ARF^+/+) and null for ARF (ARF^−/−), were injected with BrdU 20 min prior to sacrifice. Skin samples were immunohistochemically stained for BrdU incorporation, and the percentage of interfollicular basal layer keratinocytes staining positive was determined. The average percentage of cells that were BrdU positive from at least four independent mice in each group is presented.

E2F1 induces the phosphorylation of p53. (A) Primary MEF cultures wild type for ARF (lanes 1 and 2) or null for ARF (lanes 3 and 4) were coinfected with Adp53 (MOI of 25) and either AdGFP (lanes 1 and 3; MOI, 50) or AdE2F1 (lanes 2 and 4; MOI, 50). At 24 h postinfection, lysates were made and Western blot analysis was performed using antibodies specific for E2F1, p53, phosphoserine 15 p53, phosphoserine 20 p53, phosphoserine 392 p53, or ras where indicated. (B) Western blot analysis was performed on epidermal lysates (100 μg) from mice that were nontransgenic (lanes 1 and 2) or K5 E2F1 transgenic (lanes 3 and 4) and either wild type (lanes 1 and 3) or null (lanes 2 and 4) for ARF by using an antibody specific for the phosphoserine 15 form of p53. NS, nonspecific band. (C) Primary MEFs, either wild type for ARF (lanes 1 to 6) or null for ARF (lanes 7 to 12), were infected with the following recombinant adenoviruses: lanes 1 and 7, AdGFP (MOI, 200); lanes 2 and 8, AdE2F1 (MOI, 50) plus Adp53 (MOI, 25); lanes 3 and 9, Adp53 (MOI, 25); lanes 4 and 10, Adp53 (MOI, 50); lanes 5 and 11, Adp53 (MOI, 100); lanes 6 and 12, Adp53 (MOI, 200). At 24 h following infection, cell lysates were made and Western blot analysis was performed using antibodies specific for p53 (top panel), the serine 20-phosphorylated form of p53 (middle panel), or β-actin (bottom panel).