TIF-90 regulates pre-rRNA synthesis. (A) Colocalization of TIF-FL and TIF-90 with Pol I and rDNA using immunostaining and fluorescence in situ hybridization assays. The 293T cells were transfected with Myc-TIF-FL or Myc-TIF-90 and costained with anti-Myc and anti-Pol I antibodies (left). rDNA was labeled with an rDNA probe as described in “Materials and methods” (right). Fluorescence intensity was measured along the line through three-dimensional pictures on the left. (B) Interaction of TIF-FL and TIF-90 with Pol I. Interaction of glutathione S-transferase (GST)-tagged TIF-FL and TIF-90 with Pol I in cell lysate (left). Coimmunoprecipitation of Pol I and Myc-TIF-FL or Myc-TIF-90 in transfected 293T cells (right). (C and D) Effects of TIF-FL and TIF-90 expression on rRNA synthesis in primary AML cells. Primary AML cell samples (n = 10) were combined and transfected with siSCR or siRNAs specific for TIF-FL or TIF-90 for 24 hours. (C) Levels of pre-rRNA expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample as determined by qPCR and RNA synthesis using [³²P] labeling. (D) Effect of TIF depletion on rDNA promoter occupancy by Pol I. ChIP assays were performed as described in “Materials and methods” using anti-Pol I antibody. Values represent the mean ± standard deviation (SD) of triplicate determinations (n = 3) (left). The levels of expression of TIF-IA in corresponding samples in panel C are shown by western blot (right).