Skip to main content
. Author manuscript; available in PMC: 2010 Aug 25.
Published in final edited form as: Oncogene. 2009 Nov 23;29(8):1214–1226. doi: 10.1038/onc.2009.419

Figure 7.

Figure 7

Constitutively active AKT establishes cyclin D1 expression and proliferation in MCA without serum in an EGFR-dependent manner. (A) Monolayer HSC-3 cells were infected with adenovirus for GFP, KD-AKT (GFP-tagged kinase dead AKT), and CA-AKT (GFP-tagged constitutively active AKT) for 18 h. Cells were detached and subjected to MCA formation in DMEM either with or without serum (10% FBS). After 24 h, MCAs were collected, lysed, and analyzed by immunoblotting as indicated. Grb2 was used as an internal loading control. The top panel represents relative fold change of cyclin D1 as measured by NIH Image. Lanes 1, MCA with no serum; lanes 2, MCA with serum; lanes 3, MCA with serum treated with 1 μM PD168393; and lanes 4, MCA with serum and treated with 1 μM U0126. (B) GFP and CA-AKT expressing MCAs as in (A) and treated with 10 μM U0126 was lysed and analyzed by immunoblotting as indicated (C) GFP and CA-AKT expressing MCA were cultured in the presence of 50 μM BrdU with or without serum for 24 h. MCAs were then collected, trypsinized, washed, and allowed to attach on poly-lysine coated coverslips for anti-BrdU immunostaining. The total cell number and BrdU positive cells from 5–10 random microscopic fields comprising from 3–5 sample slides (* p < 0.05) were counted and represented as % BrdU incorporation (mean±SD). Results are representative of two independent experiments.