Fig. 4.

Proapoptotic tumor suppressor function of E2f2. (A) Western blot analysis of T cells derived from EμSR-tTA;Teto-MYC;E2f2^−/− tumors infected with MSCV-IRES-EGFP empty vector (con) or MSCV-E2F2-IRES-EGFP (E2F2) using anti-E2f2 antibody. Tubulin served as loading control. (B) FACS analysis of unselected cells infected with the indicated retroviruses before injection into nude mice (Upper). Representative examples of FACS analysis of individual tumors that developed in nude mice (Lower). The percentage of GFP-positive and -negative cells is indicated within the FACS diagrams. (C) Analysis of tumors that developed in mice injected with EμSR-tTA;Teto-MYC;E2f2^−/− tumor T cells that were infected either with control or E2F2 retroviruses. The data are presented as the average ratio of percent of GFP-positive cells in each individual tumor relative to the percent of GFP-positive cells before injection. n indicates a number of tumors analyzed for each group. (D) In vitro proliferation assay of unselected cells infected with the indicated retroviral constructs. Cells were plated at a concentration of 0.2 × 10⁶ per ml (day 0) and counted every 24 h for 7 days. (E) Cells were treated as in D, and the percentage of GFP-positive cells was determined by FACS. The infection efficiency at day 0 (28% for the MSCV-IRES-EGFP vector control and 39% for MSCV-E2F2-IRES-EGFP) was set to 100%. The values obtained for percentage of GFP-positive cells at each time point were plotted relative to the percentage at day 0. (F and G) The percentage of BrdU- and Annexin V-positive cells infected with the indicated retroviruses was measured 2 and 4 days after infection. For D–G, representative examples from three independent experiments are shown.