Snail c15d3

For Western blotting 20ug of exosome or cell lysate protein were analyzed by 10% SDS PAGE. Protein concentration was determined using a microBCA kit (Pierce) as per the manufacturer’s recommendations. Alix (3A9) (Cell Signaling Technology, Cat # 2171, 1:1000 dilution), GAPDH (Santa Cruz Biotechnology, Cat # sc-32233, 1:200 dilution), Snail (C15D3) (Cell Signaling Technology, Cat # 3879, 1:1000 dilution), ZEB1 (H-102) (Santa Cruz Biotechnology, Cat #: Sc-25388, 1:200 dilution), and Beta-Tubulin (Cell Signaling Technology, Cat # 2128, 1:1000 dilution). Anti-Rabbit IgG-horseradish peroxidase, anti-goat IgG-horseradish peroxidase and anti-mouse IgG-horseradish peroxidase were used as secondary antibodies (Santa Cruz Biotechnology). Chemiluminescent detection was performed using Super Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) as per the manufacturer’s recommendations. Image capture was performed using a ChemiDoc imaging system (Bio-Rad).

After transfection or treatment at the indicated concentrations and time points, cells were harvested in lysis buffer (150 mm NaCl, 50 mm HEPES, 10% glycerol, 1 mm EDTA, 0.5 mm Na₃VO₄, 10 mm NaF, 1% Triton X100, 1.5 mm MgCl₂). Protein concentrations were determined using the BCA Protein assay (BioRad). Proteins were separated by SDS/PAGE and blotted onto PVDF membranes, and immunodetection was performed with the Clarity Western ECL Substrate kit (BioRad) using the following antibodies: YB‐1 (ab12148, Abcam, Cambridge, UK, 1 : 1000); EGFR (sc‐03‐G, Santa Cruz Biotechnology, 1 : 1000); snail (C15D3, Cell Signaling Technology, 1 : 1000); and Beta‐actin (A5441, Sigma, 1 : 2000). Secondary antibodies were purchased from Dako and used at a dilution of 1 : 10 000.

The different groups of cells were harvested and lysed in RIPA buffer, followed by centrifugation. After quantification of protein concentrations using a BCA protein assay kit, individual cell lysates (30 µg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% gels, and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% fat‐free dry milk in TBST and probed with primary antibodies against E‐cadherin (4A2, 1:500), MMP2 (D4M2N, 1:1000), MMP9 (D6O3H, 1:500), N‐cadherin (D4R1H, 1:500), Slug (C19G7, 1:1000), Snail (C15D3, 1:500), Vimentin (D21H3, 1:1000), and GAPDH (D16H11, 1:1000; Cell Signaling Technology). The bound antibodies were detected with horseradish peroxidase (HRP)‐conjugated secondary antibody (Cell Signaling Technology) and visualized using the enhanced chemiluminescence kit. The data were analyzed using Image J software.

The HCC cell lines HepG2 and Huh7 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in an atmosphere containing 5% CO₂ atmosphere. Antibodies against E-cadherin (24E10), N-cadherin, claudin-1 (D5H1D) and Snail (C15D3) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against HIF-1α (GTX 127309), FOXA2 (GTX 84485), vimentin (GTX 100619) and Slug (GTX 121924) were purchased from GeneTex (Irvine, CA). Antibodies against β-actin were purchased from Sigma (St. Louis, MO, USA), and appropriate secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A commercially produced si-CPS1-IT1 (cat. no. 4390771) and negative control siRNA (cat. no. 4464058) were purchased from Thermo Fisher Scientific.

SMAD2 (L16D3; #3103) and Snail (C15D3; #3879) antibodies were from Cell Signaling Technology. Acetylated α-tubulin (clone 6-11B-1; #T6793) antibody and Phalloidin-Atto 594 (#51927) were from Sigma-Aldrich. Antibody against α-tubulin (DM1A; #CP06) was from Calbiochem, Merck Millipore. Antibodies against p-SMAD2 and p-SMAD3 have been described previously [28 (link), 29 (link)]. Goat anti-Rabbit IgG (H + L) Alexa Fluor 488 conjugate (#A-11008), Goat anti-Mouse IgG (H + L) Alexa Fluor 488 conjugate (#A-11029), and Goat anti-Mouse IgG (H + L) Alexa Fluor 594 conjugate (A-11032) were from Life Technologies. Goat-anti-Rabbit IRDye 800CW (#926-32211) and Goat-anti-Mouse IRDye 680 (#926-32220) were from LI-COR Biosciences.

The antibody against RIP1(H-207) used in immunoprecipitation and antibodies against cIAP1(F-4), cIAP2(H-85), p-ERK(E-4), JNK(D-2), p38(A-12), p-p38(E-1), and GAPDH(6C5) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against RIP1, phospho-IκBα, IκBα, and PARP used in western blotting were from BD Biosciences (Marrickville, NSW, Australia). Antibodies against p44/42 MAPK, K48-linkage Specific Polyubiquitin(D9D5), Phopho-SAPK/JNK(Thr183/Tyr185)(81E11), FLIP(D16A8), Phospho-NF-κB p65 (Ser536) and SNAIL(C15D3) were purchased from Cell Signaling Technology (Beverly, MA). Antibody against CYLD was purchased from Abcam(Melbourne, VIC, Australia). The antibody against K63-linked ubiquitin was from Novus Biologicals (Littleton, CO). Antibodies against caspase8 and caspase3 were from Enzo life sciences. The RIP1 kinase inhibitor necrostatin-1 (Nec-1), the Smac mimetic SM406, the caspase inhibitor z-VAD-fmk, the RAF Inhibitor Vemurafenib, the MEK inhibitor Trametinib, and the selective ERK inhibitor SCH772984 were from Selleckchem (Houston, TX). The NF-κB inhibitors PS1145 and BAY-11-7082, the Doxcyclin, the Cycloheximide(CHX), and the MG132 were from Sigma-Aldrich (Castle Hill, NSW, Australia). The NF-κB reporter assay kit was from QIAGEN (Valencia, CA). FITC Annexin V Apoptosis Detection Kit was from BD bioscience (Marrickville, NSW, Australia).