Sc 81417

Total protein was extracted in cells or kidney tissue using RIPA (Beyotime Biotechnology, Shanghai, China) and quantified using the BCA (Thermo Scientific, Waltham, MA, USA) assay kit. Equal amounts of protein from each sample were electrophoresed on 10% Tris-glycine sodium dodecyl sulfate-polyacrylamide gels, and western blots were transferred to 0.45-mm polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Membranes were blocked with TBST containing 5% nonfat dry milk for one hour at room temperature and then incubated with primary antibodies against mouse fibronectin (ab45688, ABCAm), vimentin (5741 P, CST), N-cadherin (22018-1-AP, ProteinTech), β-catenin (ab32572, ABCAm), snail (3879 P, CST), twist (sc-81417, Santa Cruz) or GAPDH (#5174, CST) overnight at 4 °C, followed by incubating with a horseradish peroxidase-conjugated secondary antibody. Densitometric analysis results were standardized as GAPDH expression and expressed as fold change relative to those of negative control.

Cell lysates for Western blot were collected in RIPA lysis buffer containing protease inhibitor cocktail (Thermo, 78443) for 30 min on ice and centrifuged at 13,200g for 10 min at 4°C to obtain supernatant. Protein quantification was performed with the Bradford Protein Assay Kit (Thermo, 23236). For immunoprecipitation of targeted proteins, the supernatants of collected samples were incubated with the corresponding antibody and protein A/G magnetic beads or with anti‐DYDDDDK (MBL, M185‐11) and anti‐HA (MBL, M180‐11) magnetic bead antibodies at 4°C overnight. Then, the beads were separated by a magnetic device. Cell proteins were separated by SDS‐PAGE electrophoresis followed by transfer to PVDF membranes (Millipore, ISEQ00010). After blocking with 5% skim milk (BD Difco, 232100) in Tris‐buffered saline containing 0.5% Tween 20 for 1 h, the membranes were incubated with the corresponding antibodies overnight. The targeted proteins were visualized by an enhanced chemiluminescence (ECL) detection kit (NCM Biotech, P10300) after incubation with horseradish peroxidase‐conjugated antibodies. The primary antibodies included HK2 (Abcam, ab104836), TJP1 (Abcam, ab216880), E‐cadherin (CST, 3195), vimentin (CST, 5741), Twist1 (Abcam, ab175430 and Santa Cruz, sc‐81417), β‐actin (CST, 4970S), anti‐Flag (CST, 14793), anti‐HA (CST, 3724S) and ubiquitin (CST, 3936T).

CTCs were fixed in 4% paraformaldehyde, permeabilized with phosphate buffered saline (PBS) plus 0.2% Triton X-100 and blocked for 60 min with 1% bovine serum albumin in PBS. Mouse anti-human pan-cytokeratin (C11) Ab (1:50, sc-8018, Santa Cruz Biotechnology, Heidelberg, Germany), anti-human CD45 Ab (1:50, #13917, Cell Signaling Technology, Danvers, MA, USA), anti-human TWIST1 Ab (1:50, sc-81417, Santa Cruz Biotechnology), anti-human EGFR (1:50, sc-373746, Santa Cruz Biotechnology), anti-human TRPM4 (1:50, HPA041169, Sigma Aldrich, St. Louis, MO, USA), and anti-human ZEB1 (1:50, sc-515797, Santa Cruz Biotechnology), followed by corresponding IgG Abs (Alexa Fluor^® 594, 1:100, Abcam, Cambridge, UK) were used to stain the CTCs. PureBluTM DAPI (#1351303, BioRad) labeled the nuclei. C2 Plus confocal laser scanning microscope (Nikon Instruments, Firenze, Italy) and NIS Element Imaging Software (Nikon Instruments) were used for the acquisition and processing of data. Epithelial CTCs required having a DAPI-positive nucleus with a diameter of 4 μm, cytokeratin staining surrounding 50% of the nucleus, and no CD45 expression.