Figure 7
Figure 7. Additive effects of MPA and sotrastaurin in inhibiting T-cell activation. (A) Effects of Ebp1 mutated to alanine at the S360 phosphorylation site on the regulation of rRNA synthesis. Interaction of TIF-IA with Ebp1–WT, Ebp1–S360A, and Ebp1–S360D. Jurkat T cells were co-transfected with the indicated constructs of GFP–Ebp1 and Myc–TIF-IA. Lysates were incubated with anti-Myc antibody and the precipitate immunoblotted with anti-GFP antibody (left). (Middle and right) Jurkat T cells were transfected with indicated constructs of Ebp1. 5′ETS pre-rRNA and RNA labeling (middle) and Pol I binding (right). (B) Effect of S360A–Ebp1 and S360D–Ebp1 expression on TIF-IA nucleolar localization. Jurkat T cells were transfected with WT–Ebp1, or S360A- and S360D-mutated Ebp1 constructs for 24 hours. Nucleoli were isolated and western blots were performed on nucleolar or whole cell lysate with the antibodies indicated. (C) Effects of PKCδ depletion on rRNA synthesis in T cells. Cells were transfected with SCR or siPKCδ (50 nM) for 36 hours. 5′ETS pre-rRNA levels (left), Pol I binding (middle), and PKCδ expression by western blot (right). (D) Effects of sotrastaurin on rRNA levels in T cells. The cells were treated with DMSO or sotrastaurin (100 nM) for 3 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding assay (right). (E) Effects of combined treatment with MPA and sotrastaurin on rRNA synthesis, PCNA mRNA levels, and proliferation in T cells. Cultured T cells were treated with DMSO, MPA (100 nM), sotrastaurin (100 nM), or both for 24 hours. 5′ETS pre-rRNA levels (far left), PCNA mRNA level (near left), MTS assay (near right), and western blot (far right). (F) Effects of combined treatment with MPA and sotrastaurin on rRNA synthesis and proliferation with overexpression of TIF-IA and Ebp1. Jurkat T cells were co-transfected with Ebp1 and TIF-IA for 24 hours and then treated as shown. 5′ETS pre-rRNA levels (far left), PCNA mRNA level (near left), MTS and colony forming assay (near right), and western blot (far right). (G) Inhibition of IL-2 secretion by T cells with depletion of Ebp1 and TIF-IA (left) or with treatment with MPA and sotrastaurin (right). Cells were co-transfected with Ebp1 or TIF-IA siRNA (left) or treated with indicated drugs for 24 hours (right). IL-2 levels were measured by enzyme-linked immunosorbent assay. The samples were run in triplicate. (H) Schematic model of the effects of TIF-IA and Ebp1 and of MPA and sotrastaurin on the regulation of rRNA synthesis during T-cell activation.

Additive effects of MPA and sotrastaurin in inhibiting T-cell activation. (A) Effects of Ebp1 mutated to alanine at the S360 phosphorylation site on the regulation of rRNA synthesis. Interaction of TIF-IA with Ebp1–WT, Ebp1–S360A, and Ebp1–S360D. Jurkat T cells were co-transfected with the indicated constructs of GFP–Ebp1 and Myc–TIF-IA. Lysates were incubated with anti-Myc antibody and the precipitate immunoblotted with anti-GFP antibody (left). (Middle and right) Jurkat T cells were transfected with indicated constructs of Ebp1. 5′ETS pre-rRNA and RNA labeling (middle) and Pol I binding (right). (B) Effect of S360A–Ebp1 and S360D–Ebp1 expression on TIF-IA nucleolar localization. Jurkat T cells were transfected with WT–Ebp1, or S360A- and S360D-mutated Ebp1 constructs for 24 hours. Nucleoli were isolated and western blots were performed on nucleolar or whole cell lysate with the antibodies indicated. (C) Effects of PKCδ depletion on rRNA synthesis in T cells. Cells were transfected with SCR or siPKCδ (50 nM) for 36 hours. 5′ETS pre-rRNA levels (left), Pol I binding (middle), and PKCδ expression by western blot (right). (D) Effects of sotrastaurin on rRNA levels in T cells. The cells were treated with DMSO or sotrastaurin (100 nM) for 3 hours. 5′ETS pre-rRNA and RNA labeling (left) and Pol I binding assay (right). (E) Effects of combined treatment with MPA and sotrastaurin on rRNA synthesis, PCNA mRNA levels, and proliferation in T cells. Cultured T cells were treated with DMSO, MPA (100 nM), sotrastaurin (100 nM), or both for 24 hours. 5′ETS pre-rRNA levels (far left), PCNA mRNA level (near left), MTS assay (near right), and western blot (far right). (F) Effects of combined treatment with MPA and sotrastaurin on rRNA synthesis and proliferation with overexpression of TIF-IA and Ebp1. Jurkat T cells were co-transfected with Ebp1 and TIF-IA for 24 hours and then treated as shown. 5′ETS pre-rRNA levels (far left), PCNA mRNA level (near left), MTS and colony forming assay (near right), and western blot (far right). (G) Inhibition of IL-2 secretion by T cells with depletion of Ebp1 and TIF-IA (left) or with treatment with MPA and sotrastaurin (right). Cells were co-transfected with Ebp1 or TIF-IA siRNA (left) or treated with indicated drugs for 24 hours (right). IL-2 levels were measured by enzyme-linked immunosorbent assay. The samples were run in triplicate. (H) Schematic model of the effects of TIF-IA and Ebp1 and of MPA and sotrastaurin on the regulation of rRNA synthesis during T-cell activation.

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