Ndrg2

For preparing total cell lysates, cells were lysed in lysis buffer (Invitrogen), incubated on ice for 30 min and centrifuged for 20 min to remove cell debris. Total cell lysate was subjected to SDS–polyacrylamide gel electrophoresis. The proteins were then electro-transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA) and incubated overnight with antibodies at 4°C. Subsequently, the membranes were incubated with secondary antibodies for 1 hour at room temperature and the signal was detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). The primary antibodies: NDRG2, E-cadherin, N-cadherin and vimentin were purchased from Abcam (Cambridge, MA). TWIST1 and gp-130 were purchased from Santa Cruz Biotechnology (Dallas, Texas). STAT3, p-STAT3 (Tyr705), ERK1/2 and p-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA). Flag and β-actin was purchased from Sigma. The secondary antibodies, anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology.

For nuclear and cytoplasmic extraction, cells were cultured in an RPMI-1640 medium with 10% FCS for 48 h, and then harvested with trypsin, and washed twice with PBS. Nuclear and cytoplasmic fractions were separated using the NE-PER^TM Nuclear and cytoplasmic extraction kit (Thermo, Rockford, NJ, USA) as described by the manufacturer. Equal amounts of whole cell, nuclear, or cytoplasmic lysis were loaded onto a 10% SDS-polyacrylamide gel and assayed by enhanced chemiluminescence as described by the manufacturer (PerkinElmer Inc, Waltham, MA, USA). Blotting membranes were probed with antiserum of MT3 (Sigma-Aldrich Co.), heme oxygenase-1 (HO-1; Stressgen, Victoria, BC, Canada), NDRG1 (Invitrogen), NDRG2, NDRG3 (Abcam, Cambridge, UK), HIF-1α, MASPIN (BD Biosciences, San Jose, CA, USA), HIF-2α (Novus, Littleton, CO, USA), or β-actin antiserum (Millipore, Billerica, MA, USA). The intensities of different bands were analyzed using the GeneTools of ChemiGenius (Syngene, Cambridge, UK).