Fig. 5. Thrombin stimulates both growth and contractibility through activation of the PI3K/Akt pathway. (A) Percentages of wild-type and PN-1^–/– (KO) cells incorporating BrdU (10 µM, 2 hours). The PI3K inhibitor LY294002 (LY; 50 µM) and the mTOR inhibitor RAD001 (RAD; 20 nM) were added in 10% FCS medium 24 hours before BrdU incorporation. ^***Significant difference from wild-type cells (P<0.001). (B) Volume increase of cells switched to 1% FCS medium and arrested with aphidicolin (1 µg/ml, as in Fig. 3) determined after 24 hours with or without LY294002 or RAD001. ^*Significant difference from wild-type cells (P<0.05). (C) Effects of LY294002 or GF103203X (5 µM) on thrombin-stimulated (1 U/ml) collagen gel contraction by wild-type and PN-1^–/– (KO) DP cells pretreated with hirudin (100 µM, 6 hours) and cultured in the presence of hirudin and aphidicolin. ^*Significant difference from wild-type cells (P<0.05). (D) Relative levels of phospho-Akt and total Akt proteins in extracts from wild-type and PN-1^–/– DP cells assessed by immunoblotting with the indicated antibodies. Cells were either untreated (/) or cultured in the presence of LY294002 or RAD001 as in A. (E) Effect of thrombin on Akt phosphorylation examined by adding thrombin (1 U/ml, 30 minutes) to wild-type DP cells grown in medium containing 1% FCS. The dependence of the thrombin effect on PI3K pathway was assayed by adding LY294002 or RAD001 1 hour prior to thrombin. (F) Involvement of PAR-1 in Akt activation. Wild-type DP cells were treated with thrombin (as in E), TRAP or control peptides (as in Fig. 4). (G) Comparison of the efficiency of thrombin stimulation of Akt phosphorylation in wild-type versus PN-1^–/– DP cells tested as in D. (H) Phosphorylation status of proteins of the PI3K pathway in lysates from dissected anagen whisker follicles of 20-day-old wild-type and PN-1^–/– littermates.