Figure 9
Effects of ectopic JDP2 and siRNA specific for JDP2 on proliferation and expression of cyclin-A2. (a) Reduced proliferation of WT and Jdp2KO MEFs upon expression of the JDP2 expression vector. 2 × 104 WT and Jdp2KO MEFs were transfected with 600 ng of pcDNA4-JDP2 or pcDNA and cells were counted 3 days later by the Alamar Blue method. The relative cell number on day 0 is given as one. The means of the results of three experiments±s.d. are shown. (b) Depressed cyclin-A2 reporter activity upon introduction of the JDP2 expression vector. We introduced 600 ng of pJDP2 or pcDNA4, and 400 ng of pA2M or pA2mAP1 into 5 × 104 Jdp2KO MEFs, incubated the cells for 30 h and then measured luciferase activities. (c) Depressed proliferation of MEFs upon infection with recombinant adenovirus encoding JDP2 (Adeno-JDP2). WT and Jdp2KO MEFs (2 × 104) were infected with Ad-JDP2 or β-galactosidase-encoding cDNA (Ad-cont) at a multiplicity of infection of 1 and 10. Cells growth was counted 5 days later. (d) Depressed expression of mRNA for cyclin-A2 in Jdp2KO MEFs infected with Ad-JDP2. The culture and infection of cells are described in the legend to panel c. The expression of mRNA specific for cyclin-A2 was quantitated by real-time PCR as described in the legend to Figure 7a. (e, f) Effects of siRNA specific for JDP2 on cell proliferation and expression of cyclin-A2. WT MEFs (5 × 105 cells) were transfected with 30 pmol of siRNA specific for JDP2 (JDP2si#1 and #2) and non-specific double-stranded RNA (CONTsi). Three days after transfection, cells were harvested and counted (e), and total RNA was subjected to real-time PCR using primers specific for JDP2 or cyclin-A2 (f). The levels of expression were normalized by reference to that of GPDH mRNA. Data are shown as percentages, relative to results for WT MEFs as negative control. JDP2, Jun dimerization protein-2; KO, Jdp2-knockout; MEF, murine embryonic fibroblast; siRNA, small interfering RNA; WT, wild type.
