Effects of MPA treatment on TIF-IA–regulated rRNA synthesis in T cells. (A) Expression of TIF-IA, PCNA, and Ebp1 protein in T cells stimulated for 5 days with anti-CD3 and anti-CD28. Primary T cells were isolated from PBMCs and cultured in control medium or medium containing anti-CD3/CD28. (B) Effects of TIF-IA depletion on rRNA synthesis and proliferation in primary T cells. Cells were cultured in proliferation medium and then transfected with SCR or siTIF-IA (20 nM) for 36 hours. RNA was extracted for measurement of 5′ETS pre-rRNA (left), Pol I recruitment to the rDNA promoter (middle), and MTS assay and western blot were performed as shown (right). (C) Effects of MPA on TIF-IA regulation of rRNA synthesis. Primary T cells were cultured in proliferation medium and treated with MPA for the indicated times. TIF-IA was immunoprecipitated from cell lysate and blots were probed with anti-Pol I antibody (left), ChIP assay with Pol I antibody (middle), and 5′ETS pre-rRNA levels and RNA labeling with [³²P] (right). (D) Effects of MPA on TIF-IA binding with Pol I in cells from individuals treated with MPA. TIF-IA was immunoprecipitated from cell lysate that was pooled from healthy donors (n = 6) or individuals treated with MPA (n = 5), and blots were probed with anti-Pol I antibody. (E) Effects of TIF-IA depletion on rRNA synthesis and cell proliferation in T cells treated with MPA. The cells were transfected with SCR or siTIF-IA (20 nM) for 24 hours, then treated with MPA (100 nM) for 3 hours (left and middle) or 24 hours (right). 5′ETS pre-rRNA (left), ChIP assay (middle), and MTS and western blot (right).