Bradford method

The whole cell protein was extracted using RIPA buffer combined with protease inhibitor (Merck Millipore). The protein concentration was determined by the Bradford method (Beyotime, Shanghai, China), and 20 μg of protein was separated by 10% SDS/PAGE (Beyotime) and transferred to polyvinylidene fluoride membrane (Merck Millipore). Then, the primary antibodies were incubated at 4 °C overnight and secondary antibodies for 1 h at room temperature. The primary antibodies used were mouse anti‐β‐actin (Sigma, USA), rabbit anti‐SIX1 (Proteintech, Rosemont, IL, USA), PCNA, c‐myc, cyclin‐D1 and cyclin‐A1 (Cell Signaling Technology, USA).

Western blot analysis was performed as previously described, but with some modifications (Qin et al., 2004 (link); Qin et al., 2007 (link)). Briefly, cells were lysed on ice by using RIPA lysis buffer (Beyotime, China) containing a protease inhibitor cocktail and 1% phenylmethylsulfonyl fluoride (PMSF). Total protein concentrations of the supernatants were determined using the Bradford method (Beyotime). Subsequently, proteins were separated using 12.5% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto PVDF membranes (Millipore, USA) by using a Trans-Blot apparatus (Bio-Rad). The proteins of interest were identified using the indicated primary antibodies, and detected visually using horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies. Immunoreactivity was visualized by enhanced chemiluminescence technique using SuperSignal West Pico chemiluminescent substrate (Thermo, USA).

Total protein was harvested and extracted from NSCLC tissue samples and cell lines (SPC-A1 and A549) with RIPA buffer (Millipore, Bedford, MA, U.S.A.) and the protein concentration was calculated with reagent kit via Bradford method (Beyotime, Shanghai, China). Then, protein lysates were isolated via 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and then transfected onto polyvinylidene fluoride (0.45 µm, PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin (BSA; Absin Bioscience, Shanghai, China) in 1× Tris-buffered Saline Tween-20 (TBST; Absin Bioscience), and then incubated with specific diluted primary antibodies, including E-cadherin (1:1000, ab76055), Vimentin (1:1000, ab8979), N-cadherin (1:500, ab18203), and MTDH (1:800, ab45338), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, ab8245) as endogenous control, and all primary antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). After the membranes were incubated overnight at 4°C, 1× TBST was used to wash membranes for thrice, followed by covering the corresponding second antibody. Finally, protein bands were visualized using ECL Reagent (Millipore).

Total protein was harvested and extracted from CC cells with RIPA buffer (Millipore, Bedford, MA, USA) and the protein concentration was calculated with a reagent kit via the Bradford method (Beyotime, China). The extracted proteins were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore). Then, 5% skim milk powder was used to block the membranes in the room for 1 h and then the membranes were combined with primary antibodies at 4°C all night. The next day, the membranes were washed and incubated with secondary antibodies carrying HRP-conjugates. Bands were examined through an enhanced chemiluminescence kit (Thermo Fisher).

Cellular proteins were extracted using NP40 lysis buffer (Beyotime, Nantong, China) on ice for 30 min. After measuring protein concentrations using the Bradford method (Beyotime), samples were boiled for 10 min at 100 ℃ in 1× SDS protein loading buffer (Yeasen). Following standard electrophoresis and transfer, the membrane (Millipore, USA) was blocked with skimmed milk (8%) for 60 min. Next, primary antibodies (listed in Supplementary Table 3) were added to the membrane on a glass plate for overnight incubation at 4 °C. After washing three to four times with Tris-buffered saline with Tween 20(TBST) buffer, samples were incubated with the appropriate secondary antibodies for 1 h at room temperature. After washing with TBST for three times, signals of bands were detected using the Enhanced Chemiluminescent Reagent kit (New Cell & Molecular Biotech). Image J software was used to quantitative gray value of the bands.

Total 50 mg of kidney from sham-or CLP-operated mice were placed in the liquid provided with the kit and homogenized on ice. The protein content was measure with Bradford method (Beyotime Biotechnology, Shanghai, China). Caspase-3 activity levels were acquired by caspase-3 assay kit (Solarbio, Beijing, China) based on the formation of the chromophore p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA following the protocols from the manufacturer. This analysis was used to further confirm the data of immunostaining with cleaved caspase-3 antibody, and TUNEL kit.