N cadherin

MCF7 cells (2.5 × 10⁵) were seeded on 22 mm-Aclar plastic coverslips (Pro-Plastics Inc., Linden, NJ, USA) previously coated with rat-tail collagen. After treatment with NETs (500 ng/mL) for 16 h, cells were fixed with 4% paraformaldehyde diluted in PBS (pH 7.4), permeabilized with PBS containing 0.5% Triton X-100 and incubated with primary antibodies against: β-catenin (1:50, #C-2206, Sigma Chemical Co, Saint Louis, MO, USA), E-cadherin (1:50, #04-1103, Millipore, Burlington, MA, USA), fibronectin (1:50, #F-6140, Sigma Chemical Co, Saint Louis, MO, USA) or N-cadherin (1:50, #C-3865, Sigma Chemical Co, Saint Louis, MO, USA) for 1 h at 37 °C. Cells were washed and incubated for 1 h at 37 °C with secondary antibodies Alexa Fluor 546 or Alexa Fluor 488 (1:100), all purchased from Thermo Fischer Scientific. Nuclei were labeled with 0.1 μg/mL DAPI (Thermo Fisher Scientific) or NucSpot (1:500, Biotium, Hayward, CA, USA) for 5 min. Slides were mounted in ProLong Gold antifade reagent (Molecular Probes, Eugene, OR, USA) and examined in an Axiovert 100 inverted microscope (Carl Zeiss, Oberkochen, Germany). Images were acquired with an Olympus DP71 digital camera (Olympus, Shinjuku City, Japan). The overall fluorescence intensity was quantified using the ImageJ software (NIH, Bethesda, MD, USA), and results were expressed as a percentage, considering untreated cells as 100%.

The following antibodies were used: N-cadherin (clone GC4, Sigma-Aldrich C3865, 1:100 dilution), FSP1 (Abcam ab58597, 1:100 dilution), PDGFRα (R&D Systems AF1062, 1:50 dilution), Collagen I (Rockland 600–401–103–0.1, 1:150 dilution), Collagen III (Abcam ab7778, 1:150 dilution), Fibronectin (Abcam ab23750, 1:200 dilution), α-SMA (Abcam ab5694, 1:100 dilution), CD31 (Novus Biologicals NB100-2284, 1:500 dilution), EpCAM (Abcam ab92382, 1:500 dilution), Lyve-1 (Abcam ab14917, 1:500 dilution), Myf-5 (clone C-20, Santa Cruz sc-302, 1:500 dilution), CD45 (clone IBL-3/16, Abcam ab23910, 1:500 dilution), FABP4 (clone EPR3579, Abcam ab92501, 1:500 dilution), F4/80 (Abcam ab90247, 1:500 dilution), Cytokeratin 14 (clone EPR17350, Abcam ab181595, 1:200 dilution), Integrin α_v (clone EPR16800, Abcam ab179475, 1:500 dilution), α1-catenin (clone EP1793Y, Abcam ab51032, 1:500 dilution), decorin (Abcam ab175404, 1:500 dilution), DLK-1 (Abcam ab21682, 1:200 dilution) and Ki67 (clone SP6, Abcam ab16667, 1:500 dilution). Fluorophore-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (1:500 dilution). Exherin (ADH-1) was from TargetMol (T2637).

The expression of proteins was analyzed by Western blot. The cells were incubated with ABGE for 24 hours and then lysed with RIPA buffer. Protein concentrations were determined using a plate reader (Bio-Rad, USA), and equal amounts of protein were loaded. Denatured proteins (25-30ug) were electrophoresed in 4–12% or 4–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with specific antibodies. The antibodies used were listed below: MCL-1 (#94296), BCL-2 (#3498), BIM (#26184), BAX (#41162), and BAK (#6947) were purchased from Cell Signaling Technology; NOXA (#PRS2473), PUMA (#PRS3043), E-cadherin (#SAB4503751), N-cadherin (#MABT530), Slug (#PRS3959), Vimentin (#V6389), β-Actin (#A3854) were purchased from Sigma-Aldrich; p-JNK (ab176662), JNK (ab126424), p-P38 (ab178867), P38 (ab170099), p-ERK (ab201015), ERK (ab32537) were purchased from Abcam. Blot bands were visualized after incubating with the secondary antibodies and the chemiluminescent substrate.

The AGS, MKN45, HGC27, SNU1, KATOIII, Hela and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Human GCa cell lines MGC803 were purchased from China Academia Sinica (Shanghai, PR China). The GCa MGC803, AGS, MKN45, HGC27, SNU1, KATOIII cells were cultured in RPMI-1640 medium and Hela cells, HEK293T embryonic kidney cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO₂. Antibodies against the following proteins were used with source and dilution ratios indicated: ATG4B (Cell signaling, #13507, 1:1000 and Proteintech, # Cat No. 15131-1-AP, 1:1000); P62 (Sigma, #P0067, 1:1000); LC3 (Sigma, #ABC929, 1:1000; immunofluorescence 1:100); LAMP1(CST, #15665S, 1:100); Snail (CST, #4719, 1:1000); N-cadherin (CST, #4061, 1:1000); C-caspase7 (CST, #12827, 1:1000); PARP-1 (CST, #9542, 1:1000); GAPDH (CST, #2118, 1:1000); Anti-rabbit IgG Fab2 (CST, #4412s, 1:500); Anti-mouse IgG Fab2 (CST, #4409s, 1:500). AzalomycinF4a was isolated from Streptomyces solisilvae HNM30702 and verified by the NMR and HRESIMS data [34 (link)]. Rapamycin (MCE, #HY-10219); BafilomycinA1 (MCE, # HY-100558); Tioconazole (MCE, #HY-1303191 CS-2360); Acridine Orange (Sigma, #MKCD9806); FMK9a (MCE, HY-100522); DAPI (Beyotime, ON.C1005).

For protein extraction, mice in P1 were sacrificed and dissected. Protein was extracted and prepared using the Protein Extraction kit (Keygen KGP250). The following antibodies were used: β1 integrins (1:500, Sigma-Aldrich, SAB4300655), ILK (1:1000, Sigma-Aldrich, I0783), Talin (1:100, Abcam, ab71333), N-cadherin (1:1000, Sigma-Aldrich, SAB5700640), Vinculin (1:2000, Sigma-Aldrich, V4139), Akt (1:200, Cell Signaling, #9267), Phospho-Akt (1:500, Cell Signaling, #9271), Erk1/2 (1:250, Cell Signaling, #4695), P-Erk1/2 (1:800, Cell Signaling, #4370), MRF4 (1:200, Invitrogen, PA5-120,339), Myosin (1: 200; Abcam, ab264490), Titin (1:250, Novus Biologicals, NB600-1206), Desmin (1: 80; Abcam, ab32362). Western Blot was performed as described. Relative expression levels of proteins were quantified by ImageJ 1.8.0 software.

Cells were lysed in lysis buffer [0.5% Triton X-100, 20 mM Tris (pH 7.5), 2 mM MgCl₂, 1 mM dithiothreitol, 1 mM EGTA, 50 mM β-glycerophosphate, 25 mM NaF, 1 mM Na vanadate, 100 mg/ml phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche; Indianapolis, IN, USA)]. After adjusting the protein concentration, proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected to immunoblot analysis with the indicated antibodies as follows: PLK1 (Millipore, 05–844); phospho-PLK1 T210 (Cell Signaling, 5472); phospho-PLK1 S137 (Abcam, ab21738); Vimentin (Santa Cruz Biotechnology, sc7557); E-cadherin (Cell Signaling, 4065); N-cadherin (Sigma, C3865); snail (Santa Cruz Biotechnology, sc271977); slug (Cell Signaling, 9585); GAPDH (Sigma, G8795); phospho-smad2 S465/S467 (Cell Signaling, 18338); Smad2/3 (Cell Signaling, 8685); Ki-67 (Abcam, ab16667); β-actin (Sigma, A5441); and RFP (Life Technologies, R10367). Immune complexes were displayed using an Odyssey infrared imaging system (LI-COR Biosciences; Lincoln, NE, USA) (Supplementary Fig. S8). Intensity values were determined using LI-COR Odyssey software.

Cell and tissue samples were lysed using RIPA lysis buffer (Beyotime Biotechnology Co., Ltd) for 30 min. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific protein was transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat dry milk for 30 min, specific proteins were incubated with primary antibodies [ISLR, GAPDH, Snail, Twist, E-cadherin, Vimentin, N-cadherin, biglycan (BGN), matrix metalloproteinase (MMP)-2, and secreted frizzled related protein 4 (SFRP4); 1:1000, Sigma, USA] overnight at 4°C. After three washes with Tris-buffered saline with 0.1% Tween 20 detergent, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h. Finally, enhanced chemiluminescence reagents (GE Healthcare, WI, USA) were used to visualize the protein signals [27 (link)].