Figure 5. Pten loss-induced cellular senescence involves APC-CDH1.

Cellular senescence assay (A), growth curves and BrdU incorporation assay (B), and immunoblot (C) of primary Pten^lox/lox MEFs, infected with retroviral Cre and human CDH1 selected for 4 days. Error bars represent SEM from three different experiments. P value was determined by Student’s t test. (D) Real-time RT-PCR analysis of murine p16 expression was quantified. Error bars represent SEM. (EG) Cellular senescence assay (E), growth curves and BrdU incorporation assay (F) and immunoblotting (G) of primary conditional Pten^lox/lox;Cdh1^+/+, Pten^lox/lox;Cdh1^lox/+, Pten^lox/lox;Cdh1^lox/lox MEFs, infected with retroviral Cre recombinase at 4 day after selection. Error bars represent the SEM from three different experiments. Asterisk indicates nonspecific band. (H) PC3 cells were co-transfected with wild-type or destruction box mutated (DBM) Flag-Ets2, His-ubiquitin (Ub) and HA-PTEN, and treated with the proteasome inhibitor MG132 (10 μM) for 4 hr before harvesting. His-Ub-conjugated Ets2 was purified from cell lysates using Ni²⁺-NTA spin column under denaturing conditions. (I) Cellular senescence assay of primary Pten^lox/lox MEFs, infected with a retroviral combination of wild-type or Ets2(DBM), CDH1 and Cre as indicated, at 4 day after selection. Error bars represent SEM from three different experiments. “see also Figure S4”.