Genetool program

Equal quantities of cell extract were loaded onto a 10% SDS-polyacrylamide gel and analyzed using the Western lightning plus-ECL detection system (PerkinElmer, Inc, Waltham, MA, USA). Blotting membranes were probed using GDF15 antiserum (A0185; Abclonal, Cambridge, MA, USA), MASPIN antiserum (554292; BD Biosciences), NDRG1 antiserum (42-6200; Invitrogen), NDRG2 antiserum (ab169775; Abcam, Cambridge, MA, USA), NDRG3 antiserum (ab131266; Abcam), E-cadherin antiserum (1.B.54; Santa Cruz Biotechnology), N-cadherin antiserum (AJ1526a; Abgent, San Diego, CA, USA), SLUG antiserum (C19G7; Cell signaling, Danvers, MA, USA), SNAIL antiserum (ab117866; Abcam), or β-actin antiserum (I-19, Santa Cruz Biotechnology). Band intensities were recorded using the Chemi Genius II BioImaging System of Syngene (Cambridge, UK) and analyzed using the GeneTool Program of ChemiGenius (Syngene).

The lysates were obtained by lysing cells in lysis buffer containing 50 mM Tris, pH7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 0.1% SDS, 1 mM PMSF, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of total protein were subjected to 10% SDS-PAGE and blotted onto PVDF membranes (Pall, Pensacola, FL). Western blotting was performed using rabbit anti-human COL3A1 antibody (Bioss, Beijing, China). The blotting membranes were scanned using GeneSnap acquisition software (Syngene, Cambridge, UK) and band densities were quantified with the GeneTool program (Syngene, Synoptics). GAPDH were used as internal control.

To investigate clonal relatedness, PFGE was performed on all 65 available MCRPE isolates from the 33 positive samples (one to three isolates per sample), following the Centers for Disease Control and Prevention standard protocol (41 ). Briefly, overnight cultures of E. coli isolates were suspended in cell suspension buffer, and the cells were treated with proteinase K and mixed with the agarose gel solution. The gel plugs then were treated with lysis solution, and DNA in the plugs was digested with restriction enzyme XbaI (Thermo Scientific). Gel electrophoresis was undertaken using a Bio-Rad CHEF-DRIII system, with a 200 V field at an angle of 120° run for 17–20 h, incorporating Salmonella serovar Braenderup H9812 DNA as a standard. Dendrograms were created using the GeneTool program (Syngene, India) and analyzed by the GeneDirectory program (Syngene, India).