E cadherin sc 8426

Anti-α-actinin (H-2), anti-β-actin (AC-15), and anti-SP1 (E-3, sc-17824) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cleaved PARP (5625 S), anti-cleaved caspase-9 (9501P) antibodies were from Cell Signaling. Anti-CD133 (ab19898) antibody was from Abcam (Cambridge, MA). Mithramycin A (Sigma, M6891) was purchased from Sigma and was dissolved in DMSO. Accutase (AT-104) was bought from Innovative Cell Technologies. E cadherin (sc-8426) was purchased from Santa CruzBiotechnology, Inc., and α-smooth muscle actin (αSMA) from Abcam.

Cells were lysed on ice in RIPA (radio-immunoprecipitation assay) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Samples (40-μg protein per lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinyl difluoride (PVDF) membranes, and blocked with 5% bovine serum albumin (BSA). Membranes were then incubated with primary antibodies (E-cadherin sc-8426, N-cadherin sc-7939, Vimentin sc-6260, Santa Cruz; fibulin-4 ab125073, Snail ab167609, Slug ab27568, Twist ab50887, β-catenin ab32572, C-myc ab32072, Cyclin D1 ab134175, Notch1 intracellular domain (NICD) ab83232, HES 1 ab71559, HEY L ab154077, Abcam) at working dilutions of 1:1000 at 4°C overnight. On the next day, membranes were incubated with secondary antibody for 1 hour at room temperature, and blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).

Target cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor (RIPA:PMSF = 100:1). After determining the protein concentration by the Bicinchoninic Acid (BCA) method, protein samples (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) and 5% bovine serum albumin (BSA). The membranes were then incubated on a shaking bed with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab106077, Twist ab50887, Zeb 2 ab138222, PI3K ab86714, p-PI3K ab182651, AKT ab8805, P-AKT ab38449, mTOR ab32028, p-mTOR ab109268, Abcam; p-ERK (Thr202/Tyr204) #4370, ERK #9102, Cell Signaling), at a 1:2,000 dilution overnight at 4 °C. The next day, the membranes were washed three times with TBST and then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, positive labeling of the proteins on the membranes was visualized by enhanced chemiluminescence (ECL) using a Bio-Rad ECL kit (Solarbio).

Metformin was obtained from Sigma–Aldrich (Merck KGaA, Darmstadt, Germany). The antibodies used in this study were from Cell Signaling Technology (Danvers, MA; anti-AMPK (#2532) and phospho-AMPK (Thr172, #2535), anti-NRF-1 (#69432) and NRF-2 (#20733) for immunoblotting); Abcam (Cambridge, UK; anti-PGC-1α (ab191838) and TFAM (ab272885) for immunoblotting; anti-NRF-1 (ab175932) and NRF-2 (ab31163) for immunohistochemistry); Santa Cruz Biotechnology (anti-Fibronectin (sc-8422), α-smooth muscle actin (α-SMA, sc-53142) and E-cadherin (sc-8426) for immunoblotting). A TGF-β₁ enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd. (Shanghai, China). The peritoneal dialysis fluid (4.25% Dianeal) was purchased from Baxter (Guangzhou, China).

Cells were lysed on ice in RIPA (radioimmunoprecipitation assay) buffer with 1 mM PMSF (phenylmethylsulfonyl fluoride). From the cell lysate, 40 μg of total protein was loaded into the wells of the SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, along with a molecular weight marker. Electrophoresis was carried out for 1–2 h at 100 V, followed by transfer to PVDF (polyvinyl difluoride) membranes, which were then blocked with 5% BSA (bovine serum albumin). The membranes were then incubated overnight at 4 °C with primary antibodies (fibulin-3 sc-33722, E-cadherin sc-8426, N-cadherin sc-59987, Vimentin sc-6260, Santa Cruz; Snail ab167609, Slug ab27568, Twist ab50887, β-catenin ab32572, C-myc ab32072, Cyclin D1 ab134175, Abcam) at working dilutions of 1:1000. After washing the membranes thrice with TBST for 5 min each, the membranes were incubated with conjugated secondary antibody diluted to 1:1000, at room temperature for 1 h. Blots were developed using the enhanced chemiluminescence method (Pierce™ ECL Western Blotting Substrate; Thermo Fisher Scientific, Inc.).