Inhibition of rRNA synthesis and cell proliferation in T cells by MPA. (A) Effects of MPA on rRNA synthesis and cell proliferation in cultured primary T cells. Primary T cells were isolated from PBMCs of healthy donor blood and cultured in stimulation medium (see “Methods”). (Left and middle) Cells were treated with DMSO or MPA (100 nM) for 3 hours. RNA was extracted for measurement of 5′ETS pre-rRNA and RNA was labeled with [³²P] (left). ChIP assay was performed to measure the level of Pol I recruited to rDNA promoter (middle). GAPDH (left) or 10% input (middle) were used as internal controls and samples were run in triplicate. Cells were cultured in proliferation medium in the presence of DMSO or MPA (100 nM) for 24 hours. MTS assay and western blot were performed as shown (right). (B) Effects of MPA on rRNA synthesis and cell proliferation in primary T cells from patients treated with MPA. T cells were isolated from PBMCs of healthy donors (n = 6) or individuals treated with MPA (n = 5). RNA was extracted for the measurement of 5′ETS pre-rRNA (left), ChIP assay was performed to measure the level of Pol I recruited to rDNA promoter (middle), and MTS assay and western blot were performed as shown (right). Significance was determined using the Student t test. Individual values are shown in supplemental Figure 1B. CON, control; EtBr, ethidium bromide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGS, intergenic spacer; OD, optical density.