FIG. 3.

Proliferation and cell cycle progression of primary T cells is dependent on MEK1/2, JNK, and PI3K activation. (A and B) Resting CD4⁺ T cells were left untreated (a), treated with anti-CD3/CD28 (b), or pretreated with MEK1/2 inhibitor UO126 (UO) (c), JNK inhibitor SP600125 (SP) (d), P38 inhibitor SB203580 (SB) (e), or PI3K inhibitor LY294002 (LY) (f) before stimulation with anti-CD3/CD28. Cell cycle analysis was performed by flow cytometry on PI-labeled cells (A), and percentages of cells in S, G₂ and M phase were determined as means of triplicate measurements (B). Statistically significant differences are indicated by asterisks (*, P < 0.007; **, P < 0.004; ***, P < 0.006). Results are representative of more than three independent experiments using separate donors. (C) CD3/CD28 stimulation leads to G₁ progression, as demonstrated by immunoblots showing down-regulation of P27^Kip1 and synthesis of CycD3 and Cdk4. Cell lysates were prepared at different times, and equal amounts of protein (30 μg) were resolved by SDS-PAGE followed by immunoblotting with antiserum specific for p27^kip1, CycD3, or cdk4. Immunoblotting against β-actin was performed as a loading control. (D) CD3/CD28 stimulation leads to G₁/S-phase transition: immunoblotting for Rb and cyclin A proteins. Cells were treated and cultured as for panel A. Top panel, line indicates hyperphosphorylated Rb. An immunoblot against β-actin was used as a loading control.