Fig. 4.

Hpa2 induction by MG132 involves AMPK and HSF1. (A) Immunoblotting. MKN-45 (left) and SFC-7901 (right) gastric carcinoma cells were left untreated (0) or were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed to phospho-AMPK (pAMPK; upper panels), AMPK (second panels), phospho-HSF1 (pHSF1; third panels), HSF1 (fourth panels), phospho-JNK (fifth panels), JNK (sixth panels), phospho-p70S6K (pS6K; seventh panels), S6K (8 panels), and tubulin (lower panels). (B) Inhibitors. MKN-45 cells were similarly treated with vehicle (DMSO; Con) or MG132 without (MG) or with the indicated concentrations of 17-AAG (AAG) (HSP90 inhibitor), KRIBB 11 (KRBB; HSF1 inhibitor), rapamycin (Rap; mTOR inhibitor), and dorsomorphin (Dor; AMPK inhibitor). Total RNA was extracted after 24 hours and subjected to qPCR applying primers specific for Hpa2. Note that Hpa2 induction by MG132 is attenuated by inhibitors of AMPK and HSF1 (red arrows).* P= 0.0005 for MG vs Con; ** P= 0.0007 and P= 0.0004 for MG+KRBB vs MG and MG+Dor vs MG, respectively. (C) Quantitation of Hpa2 induction by MG132 without or with KRIBB 11 (upper panel) and dorsomorphin (middle panel). Hpa2 expression is similarly decreased following silencing of HSF and AMPK-beta (C, lower panel). (D) Hpa2 cells are more sensitive to stress conditions. Control (Vo) and Hpa2 overexpressing MKN-45 cells were treated with MG132 (20 µM) for the time indicated. Cell extracts were then prepared and subjected to immunoblotting applying antibodies directed against cleaved caspase 3 (upper panel), cleaved PARP (second panel) and tubulin (lower panel). Note increased levels of the apoptotic markers in Hpa2 cells. Numbers underneath each blot specify band intensity in Hpa2 cells in relation to the same time point in control (Vo) cells. (color version of figure is available online). AMPK, activated protein kinase; Hpa2, heparanase 2; HSF1, heat shock factor-1.