Fig 5. Impact of caPDK1 on the growth factor dependence of cell metabolic activity and survival.

Each indicated cell line was transduced with a rAAV (10⁴ rAAV genomes/cell) expressing mutant PDK1 under the control of the PV P4 promoter. 72 h post transduction, the cells were treated (or not) for 4 h with 0.5 μM wortmannin prior to labeling for 30 min with Mitotracker. Mitochondrial activity and cell death were measured as described in the legend of Fig. 4. The constitutively active mutant PDK1:S138E (in A9 cells PDK1:S138E and to a lesser extent PDK1:S237D) significantly (p<0,01) reconstituted metabolic activity and prevented cells from undergoing death through necrosis (indicated by astericks). Thus, PDK1:S138E (and at least in part in A9 PDK1:S237) appeared to render cell viability independent of growth factor signaling via the PI3 kinase (marked black). It should be noted that PDK1:S138E mimics PDK1phophoS135 modification detected in non-transduced NCH149 (Fig. 4A). Transduction efficiencies were checked by confocal microscopy (S7 Fig.).