Akt regulates rRNA synthesis through TIF-90 and FLNA cleavage. (A) Effects of Akt inhibition on rRNA synthesis and cell proliferation in AML cells. K562 and a mixture of primary AML cells (n = 10) were treated with AZD8055 for 3 hours (left). The 5′ETS pre-rRNA and RNA labeling were performed as shown. Values for qPCR represent the mean ± SD of triplicate determinations (n = 3). Western blot demonstrating Akt inhibition is shown in supplemental Figure 7E. K562 and a mixture of primary AML cells (n = 10) were treated with AZD8055, and cell survival and proliferation were determined by MTT assay and western blot using the proliferation markers proliferating cell nuclear antigen (PCNA) and antigen Ki-67 (Ki67) (right). (B) Effects of TIF-90 depletion on rRNA synthesis, promoter occupancy, and cell proliferation in primary AML cells. A mixture of AML cells (n = 7) with p-Akt expression levels above the mean value (as determined by densitometry on western blot; Figure 3A) was transfected with siSCR or siTIF-90 for 24 hours. The 5′ETS pre-rRNA (left), ChIP assay (middle), and MTT assay and western blot (right). (C and D) Correlation of p-Akt with cleavage of FLNA, pre-rRNA synthesis, and cell survival in primary AML cells. Twenty-one samples from AML patients were divided into low and high p-Akt expression groups, and the level of FLNA cleavage was measured based on the densitometry results from Figure 3A. (C) Correlation of p-Akt with FLNA cleavage. (D) Correlation of p-Akt with pre-rRNA synthesis (left); correlation of p-Akt and Pol I binding to rDNA (middle); MTT assay with a mixture of AML cells (low p-Akt, n = 9; high p-Akt, n = 12) (right). (E) Effects of AZD8055 on FLNA cleavage in the presence of Akt or Akt-Myr. K562 cells were transfected with the indicated Akt constructs or treated with AZD8055 or no drug. Western blots were performed as indicated (left); 5′ETS pre-rRNA was measured by qPCR (middle) and MTT and colony-forming assay (right).