Effect of glitazone treatment on cellular signaling pathways and tumors in mice. (A) Immunoblot analysis of protein extracts from the indicated cell lines exposed to DMSO (−) or 200μM rosiglitazone for 72 hours, or 100μM ciglitazone for 96 hours was performed to visualize the expression of phosphorylated AMPK (Ser172), mTOR (Ser 2448), S6K (T421/S424), or S6 (S235/236) proteins along with total S6 and β-actin as loading controls. (B) Decrease in the average tumor size in mice treated with rosiglitazone compared with those treated with methylcellulose (vehicle). Volumes of tumors in mice treated with methylcellulose as vehicle (n = 5), 30 mg/kg rosiglitazone (n = 5), or 60 mg/kg rosiglitazone (n = 5) are plotted on the y-axis versus time in days after inoculation on the x-axis. Error bars represent the SD for each group of animals. (C) Increase in phospho-AMPK (pAMPK; T172) and decrease in phospho-S6K (pS6K; T421/S424) in mouse xenograft tumors on treatment with rosiglitazone. Immunohistochemistry of mouse xenograft tumors using antibodies specific for pAMPK and pS6K is shown. No staining was observed in the absence of a specific primary antibody (no antibody). Original magnification ×400.