Snail

Protein extracts were prepared from placental tissues and cells using RIPA lysis buffer supplemented with 1% cocktail phosphatase inhibitor and 1% cocktail protease inhibitor (Bimake, Houston, USA). The lysates were then separated by SDS‐PAGE (Bio‐Rad) and transferred onto PVDF membranes (Millipore Sigma). After blocking with 5% nonfat dry milk (Bio‐Rad) in Tris buffer containing 5% Tween‐20 for 1 h at room temperature, the membranes were immunoblotted with primary antibodies against SIRT1 (#8469, Cell Signaling Technology, Boston, USA), SIRT2~7, P53 (#10442‐1, Proteintech, Wuhan, China), P21 (#SC817, Santa Cruz Biotechnology, USA), acetylated P53 (#2525, Cell Signaling Technology, Boston, USA), vimentin (#10366‐1, Proteintech, Wuhan, China), N‐cadherin (#66219‐1, Proteintech, Wuhan, China), E‐cadherin (#20874‐1, Proteintech, Wuhan, China), SNAIL (#13099‐1, Proteintech, Wuhan, China), or β‐actin (#66009‐1, Proteintech, Wuhan, China) overnight at 4°C. After rinsing with Tris buffer containing 5% Tween‐20, the membranes were then incubated with the corresponding horseradish peroxidase‐conjugated secondary antibodies (ZSGB‐BIO) at room temperature for 1 h. Immunoreactive bands were developed using an enhanced chemiluminescent substrate (Millipore Sigma, USA), and the images were captured and analyzed by a ChemiDoc XRS+system (Bio‐Rad, USA).

Whole proteins from lung tissues or cells were extracted using RIPA buffer containing protease inhibitors. Collected lysates were boiled at 95°C for 5 min, separated on 8–12% SDS-PAGE gels, and transferred to PVDF membranes. Membranes were blocked and then incubated overnight at 4°C with primary antibodies from the following sources: Fn (Abcam, USA), α-SMA (Sigma, USA), collagen-Ⅰ (Abcam, USA), Bax (CST, USA), Bcl-2 (CST, USA), cleaved-caspase 3 (Proteintech, USA), E-cadherin (Proteintech, USA), Snail (Proteintech, USA), vimentin (Proteintech, USA), p-ERK1/2 (CST, USA), ERK1/2 (CST, USA), p-Smad2 (CST, USA), α-tubulin (Proteintech, USA), GAPDH (Proteintech, USA), and TGF-β (Abcam, USA). The bands were visualized with ECL detection reagents (Thermo Fisher Scientific, USA) and quantified using ImageJ software.

Total proteins were extracted from human tissue samples or cultured cells using RIPA buffer (Solarbio, Beijing, China) containing commercially procured protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). The total protein concentration was determined using a BCA kit (Beyotime, shanghai, China). Further, proteins were separated using a 10% SDS-PAGE and then transferred onto PVDF membranes (Merck, Darmstadt, Germany). Upon transfer, membranes were blocked using five percent skimmed milk solution in PBS/Tween-20 for an hour. The membranes were incubated overnight at 4°C with a diluted solution of primary antibodies followed by washing and re-incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Zsbio, China) for 1 h at room temperature. The following commercial antibody preparations were used: antibodies for ENC1, E-cadherin, N-cadherin, Vimentin, Snail, CD44, and CD133 were purchased from the Proteintech Group (Wuhan, China); antibodies for p-JAK2 (Tyr1007/1008), JAK2, p-STAT5 (Tyr694), STAT5, p-AKT (Ser473), AKT, and SOX2 were obtained from Cell Signaling Technology (CST, United States); and antibodies for GAPDH (internal controls) were procured from Zsbio, China.

RIPA lysis buffer was used to extract the total proteins of transfected BC cells. The quantitation of protein was evaluated using Bradford assay [19 ]. After, the sample of protein (30 µg) was separated using 10% sodium dodecyl sulphate-polyacrylamide gels and transferred onto nitrocellulose membrane (PALL, Port Washington, NY, USA), followed by a blocking at RT with 5% skim milk for 2 h. Then, the membranes were incubated with the following primary antibodies: EREG (Abcam, Cambridge, MA, USA), E-cadherin (ProteinTech, Wuhan, China), N-cadherin (ProteinTech), vimentin (ProteinTech), Snail (ProteinTech), Slug (ProteinTech), and GAPDH (ProteinTech) overnight at 4 °C. After washing three times with TBST, the membranes were incubated in the secondary antibody (ProteinTech) at RT for 3 h and washed three times with TBST. The blots were developed by electrochemiluminescence (Advansta, San Jose, CA, USA) and imaged with the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA).

Western blotting was performed according to the procedures described previously [20 (link)], loading 25 μg of total protein lysate per lane. Primary antibodies were diluted at 1:1000 and secondary antibodies were diluted at 1:5000. Antibodies against the following proteins were used for the study: HDAC7 (#33418), USP10 (#8501), β-catenin (#8480), acetyl-β-catenin (Lys49; #9030), phospho-β-catenin (Thr41/Ser45; #9565), phospho-β-catenin (Ser552; #5651), phospho-β-catenin (Ser675; #4176), Cyclin E1 (#4129), E-cadherin (#14472), Slug (#9585), β-actin (#3700), Lamin B1 (#13435), and GAPDH (#5174) (CST). HDAC7(26207–1-AP), CDK1(19532–1-AP), CDK2 (10122–1-AP), CDK4 (11026–1-AP), CDK6 (14052–1-AP), Cyclin A2 (66391–1-Ig), Cyclin B1 (55004–1-AP), Cyclin D1 (60186–1-Ig), Cyclin D3 (26755–1-AP), FGF18 (60341–1-Ig), FGFR3 (66954–1-Ig), Snail (13099–1-AP), vimentin (10366–1-AP), ZEB1 (21544–1-AP), Flag (66008–3-Ig), and ubiquitin (10201–2-AP) were also used (Proteintech Group).

Total cellular proteins were extracted with radioimmunoprecipitation assay (RIPA, Beyotime Biotechnology, China) lysis buffer with phenylmethanesulfonyl fluoride (PMSF, Beyotime Biotechnology, China) and protease inhibitor (BestBio, China) at a 100:1:1 ratio. Proteins were separated by polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Millipore, USA). The membrane was incubated with primary antibodies against: SIRT1 (1:1000, 13161-1-AP, Proteintech), SIRT2 (1:1000, 19655-1-AP, Proteintech), E-cadherin (1:1000, 20874-1-AP, Proteintech), EMT Antibody Sample Kit (1:1000, 9782, Cell Signaling), MMP2 (1:1000, 40994, Cell Signaling), MMP9 (1:1000,13667, Cell Signaling), Snail (1:1000, 3879, Cell Signaling), Snail (1:1000, 13099-1-AP, Proteintech), GAPDH (1:1000, 10494-1-AP, Proteintech), β-tubulin (1:1000, 86298, Cell Signaling), Flag (1:1000, 20543-1-AP, Proteintech) and HA (1:1000, 66006-2-Ig, Proteintech). The following day, the membranes were incubated with goat anti-mouse HRP-conjugated secondary antibody (ZB-2305) or goat anti-rabbit HRP-conjugated secondary antibody (ZB-2301) for 1 h. The proteins were visualized with the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, USA).