TI - Interaction between PP4c and PEA-15 . AB - phosPHOprotein enriched in astrocytes 15 kD ( PEA-15 ) is one of the proteins that appears to be regulated , directly or indirectly , through dePHOSphorylation by PP4c [41] . In order to investigate whether PEA-15 plays a major role in mediating the apoptosis-inducing effects of PP4c , we used PEA-15-specific siRNAs to down-regulate PEA-15 and then studied the effects of modulation of PP4c expression on the viability of these cells . CEM-C7 cells were transfected with PEA-15-specific siRNAs. 48 h post-transfection , PEA-15 down-regulation was assessed by real-time RT-PCR and Western blotting ( Fig 8a and data not shown ) , and cell viability was determined . All three siRNAs were able to down-regulate PEA-15 expression to a similar extent ( 60-70% ; Fig 8a and data not shown ) . Down-regulation of PEA-15 caused a decrease in the number of viable cells and more than doubled the proportion of apoptotic cells , confirming the involvement of PEA-15 in regulating leukemic cell proliferation and apoptosis ( Fig 8c and d ) . 48 h post-transfection , control cells ( transfected with negative control siRNA ) and cells transfected with PEA-15 siRNAs were transiently transfected with pcDNA3.1-PP4c , pcDNA3.1 ( - ) siRNA or PP4c-specific siRNAs ( transfection efficiency 50-60% ) . As similar results were obtained from all three PEA-15 siRNAs , data obtained using only one PEA-15 siRNA are shown . In the cells transfected with ( - ) siRNA , over-expression of PP4c caused 50% reduction in viable cell number , confirming the results obtained earlier ( Fig 8e ) , whereas down-regulation of PP4c caused a 50-60% increase in viable cell number ( Fig 8e ) . On the other hand , cells transfected with PEA-15 siRNAs showed a decrease in viable cell number in control cells ( cells transfected with ( - ) siRNA and vector only ) ( Fig 8e ) . Over-expression of PP4c in these cells did not have any additional effect on cell viability as it was found that the viable cell number of ( - ) siRNA transfected cells was similar to that of PP4c transfected cells ( Fig 8e ) . However , PP4c-specific siRNAs partially reversed the effects of PEA-15 down-regulation ( Fig 8e ) . In order to ensure that the effects of PEA-15 down-regulation on PP4c function were specific , we analysed the effects of PP2cbeta over-expression on PEA-15 siRNA transfected cells . Cells transfected with ( - ) siRNA or PEA-15 siRNAs were transiently transfected with pCMVSPORT6-PP2cbeta or pCMVSPORT6 , and viable cell number was determined after 24 h . Fig. 8f shows that , in contrast to PP4c , transfection of PEA-15 knock-down cells with PP2cbeta led to an additional loss of viable cells , confirming that although PP4c and PP2cbeta have similar effects on cell viability , their mechanisms of action are different .