DNA damage induces E2F3a. (a) Saos2 cells were left untreated (Unt) (control) or treated with doxorubicin (DX) (300 ng/ml), etoposide (ET) (6 μg/ml), or neocarzinostatin (NCZ) (300 ng/ml) overnight. E2F expression was determined by Western blot analysis. (b) U2OS cells were treated overnight with doxorubicin (DX) or etoposide (ET) at the indicated doses. (c and d) Western blot analysis was performed to detect E2F expression. HCT116 (c) or MDAH041 (d) cells were treated with doxorubicin overnight and then harvested for Western blot analysis. The polyclonal antibody, C-18, was used to detect E2F3 expression in panels a to d. (e) Saos2 cells were transfected with different amounts of siRNA (control [CT], E2f3#1, and E2f3#2 siRNA) as indicated. Two days later, whole-cell extracts were used for Western blot analysis. Note that the N-20 antibody fails to detect any changes, whereas the C-18 antibody detects the dose-dependent siRNA-mediated knockdown of E2F3. Two different lots of the N-20 antibody failed to detect E2F3a by Western blot analysis. (f) Saos2 cells were transfected overnight with control or E2f3 siRNA. The following day, the transfected cultures were divided into three plates, allowed to recover overnight, and then treated with doxorubicin as indicated. Western blot analysis was performed with a monoclonal E2F3 antibody (PG-14) and actin.

chk-dependent phosphorylation of E2F3a promotes its posttranslational induction in response to DNA damage. (a) Saos2 cells were transfected with either a control or chk1 siRNA. The next day, the cells were split into different dishes, and 24 h later, they were treated overnight with doxorubicin. The cells were harvested, and Western blot analysis was performed with the indicated antibodies. (b) Schematic representations of E2F3a and E2F3b proteins. Note that the putative chk1 site is present only in the N terminus of E2F3a. (c) Saos2 cells were transfected with an empty vector, a vector expressing wild-type (WT) E2F3, or the E2F3-S124A mutant (carrying a serine to alanine mutation at residue 124) and a green fluorescent protein (GFP) expression vector. The next day, the cells were treated overnight with doxorubicin and then harvested for Western blot analysis. Western blot detection of GFP was used as a control for transfection and loading. To calculate the fold induction, E2F3a levels were normalized to the GFP levels and expressed relative to their respective untreated sample. Note that the S124A mutant is not induced in response to DNA damage. (d) A GST E2F3a fusion protein carrying either the wild-type or S124A mutant N terminus was used as a substrate for an in vitro kinase assay with recombinant chk1 and chk2. The results of autoradiography of the phosphorylated substrates and a Coomassie blue-stained gel demonstrating that equivalent amounts of the E2F3 substrates were used in the kinase reactions. (e) An in vitro kinase assay was performed as described above except that no radioactive label was added to the reaction mixture. Western blot analysis was performed with an anti-phospho-S124 antibody (anti-P-S124) or anti-E2F3 antibody. (f) Saos2 cells were treated with doxorubicin and harvested for Western blot analysis with the indicated antibodies. (g) Saos2 cells were transfected with a control or chk1 siRNA as in panel a, and then treated with doxorubicin. Western blot analysis was performed with the indicated antibodies.

The activating E2F transcription factors are transcriptionally induced by some DNA damaging agents. (a) Saos2 cells were treated overnight with doxorubicin (DX) (300 ng/ml), etoposide (ET) (6 μg/ml), or cisplatin (CIS) (6 μg/ml). Total RNA was isolated and used for RT-PCR analysis of E2f1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (b) Saos2 cells were treated overnight with the indicated doses of doxorubicin and total RNA was isolated and used for RT-PCR analysis to detect E2f1, E2f2, E2f3a, or GAPDH. (c) Normal IMR90 cells were treated overnight with the indicated doses of doxorubicin and then processed for RT-PCR analysis to detect E2f1 and GAPDH. (d) Normal IMR90 cells were treated with doxorubicin and then processed for real-time RT-PCR analysis of E2f1 and E2f3. (e) Control or E2f3 siRNA was transfected into Saos2 cells, and on the following day, the cells were replated, allowed to recover overnight, and then treated with doxorubicin. The next day, the cells were harvested and processed for RT-PCR analysis of E2f1, E2f2, and GAPDH. (f) Saos2 cells were treated overnight with doxorubicin. The cells were fixed and processed for chromatin immunoprecipitation (IP) analysis to detect the association of E2F3 with the E2f1 promoter.

E2f1 is not required for E2F3a induction by DNA damage. (a) Mouse embryonic fibroblasts derived from wild-type, E2f1 null (E2f1 KO) or E2f1 and E2f2 double null (E2f1/2 KO) mice were treated overnight with doxorubicin and processed for Western blot analysis of E2F3 expression with the monoclonal antibody PG-14 and actin. Note that neither deficiency in E2f1 or E2f2 affects the induction of E2f3a in response to DNA damage. (b) MEFs of the indicated genotypes were treated as described above for panel a and then processed for real-time RT-PCR to detect E2f3a expression. Similar results were obtained in another independent experiment. DKO, double knockout.

E2f3 deficiency abrogates irradiation-induced apoptosis in the developing CNS. Pregnant E2f3 heterozygous females were irradiated with 5 Gy and sacrificed 5 h later, and the embryos were processed as described in Materials and Methods. (a) Representative pictures of TUNEL staining in the developing CNSs of embryos irradiated at 13.5 dpc. Shown are pictures of an E2f3 heterozygous (HET) and knockout (KO) embryo. The black arrows point to examples of apoptotic nuclei. (b) The extent of apoptosis after irradiation was assessed in heterozygous and knockout mice. At least 300 cells were counted per field, and several fields were assessed for each embryo. A minimum of four embryos were assessed for each genotype.

DNA damage induces E2F3a in a transcriptional and posttranslational manner. Saos2 cells were treated with doxorubicin (DX) (300 ng/ml) and harvested at the indicated times (in hours). (a and b) Northern (a) or Western (b) blot analysis was performed to detect E2F3 expression. EtBr, ethidium bromide. (c) Saos2 cells were transfected with a myc-tagged E2F3 expression vector or a control vector. The next day, the cells were treated with doxorubicin and then harvested the following day for Western blot analysis with an anti-myc tag antibody. (d) Cycloheximide (CHX) was added to doxorubucin-treated Saos2 cultures, and then the cells were harvested at the indicated times (in minutes). (e) Densitometric analysis was performed using the NIH ImageJ software to determine the stability of E2F3a protein in control and doxorubicin-treated cells. Similar results were obtained in three independent experiments.

E2F3 is required for the induction of E2F1 by doxorubicin. (a) Saos2 cells were transfected with a control, E2f1, or E2f3 siRNA. The following day the cells were replated into parallel cultures and then treated with doxorubicin as indicated. Western blot analysis was performed on whole-cell extracts to detect E2F1, E2F2, E2F3, and actin. (b) Saos2 cells were transfected with a control siRNA or an siRNA targeting the 3′ untranslated region (E2f3 UTR) of E2f3. The cells were replated into parallel plates and treated as indicated with doxorubicin. Western blot analysis was performed with the indicated antibodies. Note that both E2f3 siRNAs (from panels a and b) downregulate E2F1 expression in Saos2 cells. (c) U2OS osteosarcoma cells were transfected with either a control or E2f3 UTR siRNA. The following day, the cells were replated into parallel plates and subsequently treated with doxorubicin as indicated. Western blot analysis was performed with the indicated antibodies. (d) Normal Wi38 lung fibroblasts were transfected twice with control or E2f3 siRNA. The cells were then replated into parallel cultures and subsequently treated with doxorubicin. The cells were harvested the next day and processed for Western blotting with the indicated antibodies. A monoclonal antibody (PG-37) was used to detect E2F3.

Transcriptional induction of E2f1 in response to DNA damage is E2f3 dependent. (a) Mouse embryonic fibroblasts from wild-type or E2f3 null mice were treated overnight with doxorubicin. Western blot analysis was performed to detect the expression of E2F1, E2F3a, and actin. E2F3a was detected with the monoclonal antibody PG-14. (b) MEFs treated as described above for panel a were harvested, and total RNA was used to perform real-time RT-PCR analysis of E2f1 expression. Note the absence of E2f1 induction in three different E2f3 knockout (KO) MEF cell lines. (c) E2f3 knockout cells were infected with a retrovirus carrying either an empty vector (pBabe) or myc-tagged E2f3 expression vector (myc-E2f3a). After selection, the cells were treated with doxorubicin, and Western blot analysis was performed as indicated. (d) E2f3 knockout cells treated in parallel as described above for panel c and then processed for RT-PCR analysis of E2f1 and GAPDH expression.

E2F3 mediates chemotherapy-induced apoptosis. (a) Saos2 cells were transfected with a control, E2f1, or E2f3 siRNA. Two days later, the cells were treated with etoposide (6 μg/ml), doxorubicin (300 ng/ml), or neocarzinostatin (300 ng/ml) for 48 h. The cells were fixed in culture, and nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI) to permit the identification of apoptotic (pyknotic) nuclei. (b) U2OS cells were transfected with a control or E2f3 siRNA and then replated the next day as described above for panel a. Doxorubicin (500 ng/ml) or etoposide (6 μg/ml) was added to the cultures, and the cells were harvested the following day. The apoptotic cells were assessed as described above for panel a. A minimum of 300 cells were counted in each condition, and the experiment was repeated at least three times. Similar results were obtained with two different E2f3 siRNAs. (c) Saos2 cells were transfected with the indicated siRNAs (control, E2f1, and E2f3) and harvested 48 h later for analysis of TA-p73 expression by real-time RT-PCR. (d) Saos2 cells were transfected overnight with the indicated siRNAs (control, E2f1, and E2f3), replated the following day into two plates, and allowed to recover overnight. The cells were then either left untreated (UNT) or treated with doxorubicin (DX) overnight and then harvested for real-time RT-PCR analysis of TA-p73 expression. Note that either E2F1 or E2F3 knockdown suppresses the induction of p73 by doxorubicin.