FIG. 4.

Effect of E2F4 induction on the response of differentiated PC12 cells to NGF withdrawal. (A) Western analysis of E2F protein expression following removal of NGF from wild-type PC12 cells. PC12 cells were treated with NGF in serum-containing medium for 14 days and then washed (w) and cultured with serum for up to 4 days. (B) Phase-contrast microscopy of E2F4-tet cells treated with NGF in the presence of serum for 8 days, then washed three times to remove NGF, and incubated in either the presence [wash (+)] or absence [wash (−)] of serum for 2 days, as indicated. Tetracycline was either present in (E2F4 off) or absent from (E2F4 on) the culture medium. (C) Neurite outgrowth in E2F4-tet derivatives following the washing of NGF-treated cells. Cells were treated with NGF in serum-containing DMEM for 8 days and then were washed in medium with or without serum for 2 or 4 days. Data were analyzed as for Fig. 3. (D) Western analysis of β-tubulin expression. E2F4-tet cells were treated with NGF in serum-containing medium for 8 days with or without tetracycline (tet) and then either harvested (8 days) or washed (w) and cultured in the absence of NGF for 2 or 4 days. The same membrane was probed for Sp1 protein as an internal control.