Figure 7.

(A) “Knockdown” of JAZ by siRNA increases E2F transcriptional activity and attenuates serum withdrawal-induced G₁ arrest in both p53^+/+ and p53^−/− MEFs. The JAZ-siRNA was transfected into p53^+/+ or p53^−/− MEFs. After 48 h, cells were lysed for protein gel analysis using JAZ111 and a Tubulin antibody (top part). Endogenous JAZ expression was “knocked down” by ∼70%, as determined by densitometry.¹⁴ The JAZ-siRNA was also co-transfected into the MEFs with pE2F-TA-luciferase vector and a carrier vector pEGFPN1 (GFP). After 48 h, cells were grown with or without serum for 24 h and then assayed for luciferase activity (middle part). *p < 0.01; **p < 0.001 vs. the control siRNA. In addition, the JAZ-siRNA had no effect on the activity of the pTA-luciferase control vector (data not shown). Furthermore, after siRNA transfection for 48 h followed by serum deprivation for 24 h, flow cytometry analysis was performed (bottom part). *p < 0.001; **p = 0.001 vs. the control siRNA. (B) “Knockdown” of JAZ expression accelerates serum-stimulated G₁/S progression in M1 cells. p53-deficient M1 leukemic cells in which JAZ was stably “knocked down” (Sup. Fig. 1sB) were synchronized in the G₁ phase by incubation with 1.5 mM hydroxyurea for 18 h. Cells were then washed and repleted with fresh growth media containing 10% FBS. At 0, 3, 6, 9 and 12 h following serum stimulation, cells were harvested for flow cytometry analysis (top part). The changes in G₁/G₀ (decreased percentage) at 3 h and 6 h after serum stimulation are shown (bottom part). *p = 0.008; **p = 0.003 vs. the control siRNA.