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. 2008 Jan 31;27(5):792–803. doi: 10.1038/emboj.2008.13

Figure 6.

Figure 6

Activated Ras regulates the TAp73/ΔNp73 ratio through PI3K-signalling. (A) Restoring TAp73 expression inhibits anchorage-independent growth of BJ-TER cells. BJ-TER cells were transfected with TAp73 or GFP as a control and tested for anchorage-independent growth in soft agar. Shown is the clonogenic growth in soft agar medium and the mean number of colonies±s.d. Scale bars, 200 μm. (B, C) Knockdown of ΔNp73 inhibits soft agar growth of BJ-TER cells. (B) ΔNp73 was detected by a western blot with a polyclonal antiserum raised against the ΔNp73-specific epitope encoded by the alternative exon 3′. ΔNp73-specific (◂) and non-specific (*) bands are indicated. ΔNp73 was detected under both basal and p53-stimulated conditions following expression of a non-silencing control (ns) or ΔNp73-directed shRNA. (C) BJ-TER cells were transduced with the lentiviral shRNA constructs as indicated and tested for growth in soft agar. Shown are representative images of soft agar colonies and the mean number of colonies±s.d. Scale bars, 200 μm. (D, E) Semiquantitative RT–PCR for TAp73 and ΔNp73 expression and western blot for TAp73 in H-RasV12-expressing BJ-TER cells treated with the MEK-inhibitor U0126 or the PI3K inhibitor LY294002 for 24 h. Non-transformed BJ-TE cells are shown for comparison. (F) BJ-TER cells were treated with LY294002 or DMSO as a control and tested for growth in soft agar. Shown are colony morphology and the mean number of colonies±s.d. (G) PI3K pathway activation switches the TAp73/ΔNp73 ratio in BJ-TE cells. BJ-TE cells were transduced with retroviral vectors encoding H-RasV12WT and the effector loop mutants H-RasV12T35S, H-RasV12E37G and H-RasV12Y40C. Shown are western blots for PI3K-AKT and p44/42 MAPK pathway activation. For specific detection of TAp73 and ΔNp73, we first immunoprecipitated total p73 with a polyclonal p73 antiserum before western blot detection with antibodies specific for TAp73 and ΔNp73 epitopes. (H) HCT116 cells were transduced with a non-silencing (ns) or a K-RasD13-directed shRNA with or without coexpression of H-RasV12WT or H-RasV12Y40C. PI3K-AKT and p44/42 MAPK pathway activation, TAp73 and ΔNp73 expression were analysed by western blot in HCT116 cells grown to confluence.