FIG. 4.

c-Jun/ATF2 heterodimers are necessary and sufficient for CGN apoptosis. (A) CGNs transfected with c-Jun-DN, ATF2-DN, or A-ATF2 together with GFP (to mark the transfected cells) were maintained in 25 K or 5 K medium for 12 h and stained with Hoechst 33258. (B) HEK 293 cells were cotransfected with the constructs BS/U6, shc-juna, or shc-junb together with Flag-c-Jun and subjected to Western blotting with a Flag antibody (left panel). CGNs transfected with BS/U6, shc-juna, or shc-junb plasmids were maintained in 25 K or 5 K medium and then subjected to c-Jun staining, with β-Gal staining as a marker for transfection (middle panel) or with GFP to analyze apoptosis (right panel). Scale bar = 10 μm. (C) Knockdown efficiency and apoptotic analysis for shatf2a and shatf2b were performed as described for panel B. (D) HEK 293 cells were transfected with V5-C2/c-Jun and Flag-C2/ATF2 and then lysed for CoIP with V5 or Flag antibodies. Western blotting (WB) was performed using a Flag or V5 antibody. (E) CGNs were cotransfected with C2/c-Jun and C2/ATF2 in the indicated doses and in the absence or presence of atf-luc plasmids (left panel). A dual-reporter analysis was performed. An apoptotic analysis was performed 48 h posttransfection. All apoptotic rates in this figure were quantified by scoring the percentages of transfected neurons with pyknotic nuclei in total transfected cells. The data are presented as means ± SE of the means (three experiments). *, P < 0.05; **, P < 0.01 (Student's t test).