FIG. 4.
v-Fos has no effect on proliferation or serum or anchorage dependence. (A) Telomerase-immortalized fibroblasts. Tif, Tif-puro 1, Tif-puro 2, Tif-Fos 1, and Tif-Fos 2 exhibited an increase in saturation density with each passage after 80 cumulative P.D., which correlated with a loss of p16INK4a expression as demonstrated by Western blotting after 100 P.D. hff/21 (36 P.D.) was used as a positive control for p16INK4a, and equal loading was determined with anti-ERK2. (B) Growth curves of telomerase-immortalized fibroblasts expressing v-Fos. Cells (p16+ve and p16−ve) were grown in 20% serum, and once exponential growth was established after 72 h, the cells were washed and incubated with either 20% serum or a low concentration of serum (0.2%), as indicated by the arrow. Cell proliferation was determined by counting the number of attached cells every day. Each experiment was performed in triplicate, and the error bars show the standard deviation of the mean daily count. (C) Anchorage-dependent growth of telomerase-immortalized fibroblasts with various combinations of v-Fos, loss of p16INK4a (p16−ve), and inactivation of p53 with HPV16 E6 or mutant p53 (273H). For each experiment, 104 or 105 cells were seeded in soft agar. Multiple experiments with multiple polyclonal populations of the cells were performed. During a 3-week period, colony formation was monitored and scored as 0 if there were no soft agar colonies. Tif-Ras that had lost expression of p16INK4a and expressed a mutant form of p53 (mtp53) were used as a positive control for colony formation in soft agar.