Fig. 5.

DOHH activity affects malignant transformation of 3T3 fibroblasts. (A) Population doublings of Dohh^flox/flox; Cre^pos. or Dohh^flox/flox; Cre^neg. 3T3 cells after treatment with 4-OHT or GC7, relative to untreated control cells. The 3T3 cells were retrovirally transduced with c-Myc. (B) Population doublings of Dohh^flox/flox; Cre^pos. or Dohh^flox/flox; Cre^neg. 3T3 cells after treatment with 4-OHT or GC7, relative to untreated control cells. Cells were transformed by subsequent retroviral transduction with c-Myc and H-Ras^V12. (C,D) Quantification of senescence by SA-β-Gal assay in cells described in A and B after one week of 4-OHT or GC-7 treatment. (E) 2D western blots against eIF5A1 of H-Ras^V12 and c-Myc transformed Dohh^flox/flox; Cre^pos. cells treated with 4-OHT or GC7 or with both substances. (F) Number of colonies grown in soft agar after 9 days of pre-treatment with 4-OHT/GC7 in c-Myc and H-Ras^V12 transformed Dohh^flox/flox; Cre^pos. or Dohh^flox/flox; Cre^neg. cells. GC7 was furthermore added to the top agar for the 18 days of incubation, whereas no extra 4-OHT was applied. (G) Representative photographs (left) and light microscopic images (right) of crystal violet stained and unstained colonies grown in soft agar of cells described in F. Level of significance was determined for all experiments using t-tests (***P<0.001, **P<0.01, *P<0.05). All analyses were performed at least in triplicate.