Figure 9

N-Ras regulation of Bax and PERP expression. (a) Transcriptional activation of Bax is N-Ras-dependent. Relative luciferase activity of transfected Bax construct versus their empty vector controls was measured in WT, N-ras^-/- and N-ras^-/- with partially restored N-ras expression cells as described in Materials and methods. The assays were carried out twice, each time in triplicate, with error bars indicating standard deviation (**P < 0.01; ***P < 0.001 versus control; ⁺P < 0.05; ⁺⁺P < 0.01 versus N-ras^-/- fibroblasts). (b) Transcriptional activation of Perp is N-Ras-dependent. Relative luciferase activity was measured in transfected WT, N-ras^-/- and H-ras^-/-/N-ras^-/- fibroblasts as well as in double knockout fibroblasts with partially restored expression of either H-ras or N-ras. The assays were done twice, each time in triplicate, with error bars indicating standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001 versus WT cell lines; ⁺P < 0.05; ⁺⁺P < 0.01 versus N-ras^-/- fibroblasts; Ψ P < 0.05; ΨΨ P < 0.01 versus H-ras^-/-/N-ras^-/- fibroblasts). (c) Western immunoblot showing recovery of H-Ras and N-Ras expression after transfection of double knockout H-ras^-/-/N-ras^-/- fibroblasts with vectors containing a copy of either H-ras or N-ras. (d) Regulation of Bax expression and activation through the ERK and p38 pathways. Control fibroblasts were treated with different Ras-effector inhibitors as indicated in Materials and methods, and total Bax levels were detected by immunoblot. (e) Controls of activity of the chemical inhibitors on their corresponding molecular targets.