Bio imaging system

Cells and human RCC tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China), supplemented with protease inhibitors (Roche, Shanghai, China) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Roche), at 4°C for 30min. The cell supernatants were extracted after centrifugation for 15 min at 14,000 rpm, then the concentration of the protein was determined using a BCA Protein Quantification kit (Beyotime Institute of Biotechnology). Proteins from tissues and cells were separated using 10% SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, USA), blocked for 2h with 5% nonfat milk at room temperature, and incubated with primary antibodies at 4°C overnight. After that, the membranes were washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, CA, USA), and antibodies against β-actin served as a negative control. Rabbit monoclonal antibodies (1:1000) against TGF-β1, E-cad, N-cad, Vimentin and MMP-9 (Cell Signaling Technology, USA) were used in Western blot analysis according to the manufacturer’s instructions.

Cells and human RCC tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Beijing, China), supplemented with protease inhibitors (Roche, Shanghai, China) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Roche), at 4°C for 30min. The cell supernatants were extracted after centrifugation for 15 min at 14,000 rpm, then the concentration of the protein was determined using a BCA Protein Quantifcation kit (Beyotime Institute of Biotechnology). Proteins from tissues and cells were separated using 10% SDS-PAGE, transferred onto PVDF membranes (Millipore, Billerica, USA), blocked for 2h with 5% nonfat milk at room temperature, and incubated with primary antibodies at 4 °C overnight. After that, the membranes were washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, CA, USA), and antibodies against β-tubulin served as a negative control. Rabbit monoclonal antibodies (1:1000) against TGF-β, Smad2/3, p-Smad2, p-Smad, matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 9 (MMP9) (Cell Signaling Technology, USA) were used in Western blot analysis according to the manufacturer's instructions.

The RIPA kit (Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride was used to extract protein lysate from CRC tissues and cells according to the protocols. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to quantify the protein concentration. The protein lysate was separated via 10% SDS-PAGE (Beyotime, Shanghai, China), next, it was transferred onto a polyvinylidene fluoride membrane (Millipore, USA). The membranes were blocked with 5% skim milk powder for 2 h and then incubated with the specific primary antibody overnight at 4°C, followed by a wash in TBST. Then, the membrane was incubated with the corresponding secondary antibody for 2 h at room temperature and then rinsed in TBST for three times (10 min each time). The level of protein expression was detected by ECL Plus (Millipore, USA) using a Bio-Imaging System (Bio-Rad, USA). The antibodies against SP1, IκBα, p- IκBα, p65, p-p65, E-cadherin, ZO-1, N-cadherin, Vimentin, HIF-1α, Histone H3, and β-actin were purchased from Abcam (Cambridge, MA, USA).