Genetools of chemigenius

Equal amounts of whole-cell lysate were loaded onto a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel and assayed by enhancing the chemiluminescence as described by the manufacturer (PerkinElmer Inc., Waltham, MA, USA). The blotting membranes were probed with antiserum of NIK, IĸBα, Akt, phospho-Akt^S473, Slug (Cell Signaling Technology, Danvers, MA, USA), MMP9, Snail (Abcam, Cambridge, MA, USA), MIEN1 (OriGene Technologies), NDRG1 (Invitrogen Thermo Fisher Scientific Inc., Waltham, MA, USA), E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), N-cadherin (Abgent, San Diego, CA, USA), or β-actin (Merck Millipore, Burlington, MA, USA). The intensities of different bands were analyzed using the GeneTools of ChemiGenius (Syngene, Cambridge, UK).

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).

Cells were incubated in the RPMI-1640 medium with 10% FCS and different treatments for a period of 24 hours. The nuclear and cytoplasmic fractions were extracted by NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Scientific, Rockford, IL). Equal quantities of cell extract were loaded onto a 10% sodium dodecyl sulfate polyacrylamide (SDS) gel and analyzed by the electrochemiluminescent detection system. The blotting membranes were probed with 1∶1000 diluted IκB kinase α (IKKα) antiserum, 1∶1000 NFκBp50 antiserum, 1∶1000 diluted NFκBp65 antiserum (Merck Millipore, Darmstadt, Germany), 1∶1000 diluted PARP (Cell Signaling, Danvers, MA), 1∶200 diluted NFκB-inducing kinase (NIK) antiserum, 1∶1000 diluted IκB antiserum, 1∶200 diluted Lamin B antiserum, or 1∶3000 diluted β-actin antiserum (Santa Cruz Biotechnology, Santa Cruz, CA). The intensity of different bands was recorded and analyzed by GeneTools of ChemiGenius (Syngene, Cambridge, UK).