Figure 7: p53 ISGylation suppresses cell growth and tumour development.

(a) p53^−/− HCT116 cells expressing wild-type p53 (Wt), its 2KR mutant, or an empty vector (Mock) were transfected with shNS or shISG15. After treatment with doxorubicin for increasing periods, viable cells were counted by staining with trypan blue exclusion. Error bar, ±s.d. (n=3). (b) p53^−/− HCT116 cells prepared as in a were incubated with or without doxorubicin for 10 days. The colonies were then stained with crystal violet. (c) Experiments were repeated as in b and the colonies were counted. Error bar, ±s.d. (n=3). (d) shNS or shISG15 were expressed in both p53^+/+ and p53^−/− HCT116 cells with and without shRNA-insensitive Flag-ISG15. After treatment with doxorubicin, experiments were then performed as in b. Error bar, ±s.d. (n=3). (e) p53^+/+ HCT116 cells expressing shNS or shISG15 (top) and p53^−/− HCT116 cells expressing wild-type p53 or its 2KR mutant (bottom) were injected to BALB/c nude mice. ‘Mock’ in the bottom panel indicates the cells transfected with an empty vector. After treatment with PBS or doxorubicin, tumour volumes were determined. Error bar, ±s.d. (n=5). (f) A model for positive feedback regulation of p53 transactivity by ISGylation under DNA damage conditions. ‘Ac’ and ‘P’ indicate acetylated and phosphorylated, respectively.