Ab98952

3T3-L1 cells were supplied by OTWO (Shenzhen, China). Fetal bovine Serum (FBS) and RPMI 1640 medium (1640) were obtained from Gibco (California, USA). D-(+)-Glucose Solution (20 %, sterile) was obtained from Beyotime Biotechnology (Shanghai, China). FGF21, CAY10650, and 1-Dodecylimidazole (NDI) were bought from MedChemExpress (New Jersey, US). Primary antibodies against CTSD (69854S), C-CASP8 (8592S), and β-catenin (8480S) were purchased from Cell Signaling Technology (US). Primary antibodies against P62 (ab109012) and N-cadherin (ab98952) were supplied by Abcam (Cambridge, UK). Primary antibodies against α-SMA (701457) were obtained from Thermo Fisher Scientific (Massachusetts, US). Primary antibodies against LC3B (A17424) were obtained from ABclonal (US). Primary antibodies against E-cadherin (AF0131) were bought from affinity (OH, US). Primary antibodies against α-Tubulin (R23454), β-actin (R23613), anti-mouse and anti-rabbit secondary antibodies IgG-conjugated with horseradish peroxidase (HRP) and Ultrasensitive enhanced chemiluminescence (ECL) kit were obtained from ZenBioScience (Chengdu, China). Donkey Anti-Rabbit/Mouse IgG H&L (Alexa Fluor® 488) pre-adsorbed, Donkey Anti-Rabbit/Mouse IgG H&L (Alexa Fluor® 647) pre-adsorbed were obtained from Abcam (Cambridge, UK).

According to RIPA lysis buffer (Solarbio), total proteins were prepared. Next, 30‐μg protein samples were size‐fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After transferring onto PVDF membranes (2 h, 100 V), primary antibodies (4°C, overnight) were incubated. After incubation with secondary antibody, protein bands were observed according to the enhanced chemiluminescence solution (Vazyme, Nanjing, China). Antibodies (Abcam, Cambridge, UK) contains E‐cadherin (ab231303; 1:1500), N‐cadherin (ab98952; 1:1000), HOXB7 (ab152454; 1:1500), β‐actin (ab8227; 1:2000), and secondary antibodies (ab205718/ ab205719; 1:4000). Western blot assay was conducted at least three times.

SW480 and HCT116 cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. After blocking with 10% normal goat serum, the cells were then incubated with anti-E-cadherin (1:200, ab40772, Abcam, Cambridge, UK) or anti-N-cadherin (1:200, ab98952, Abcam) antibody at 4°C overnight. Thereafter, cells were incubated with Alexa Fluro 488 or 594-conjugated secondary antibodies (2 μg/mL, A11001 or A11012, Invitrogen) and mounted in ProLong Gold Antifade Mountant with DAPI (Invitrogen). Images were photographed using a confocal microscope (Zeiss).

Lung tissue sections were deparaffinised in xylene and antigen retrieval carried out using target retrieval citrate buffer pH 6.0 (Dako S2369) for 15 min. Tissues were immunostained with polyclonal rabbit anti-human S100A4 (1:1000; Dako A5114), mouse monoclonal VE-cadherin (CD144) (1:150; Thermofisher 14144982), vimentin, monoclonal mouse (1:200; Dako M7020), mouse monoclonal anti-N-cadherin (1:100; Abcam ab98952), monoclonal mouse anti-α-SMA (1:500, M0851, Dako), collagen type I (1:200, Abcam ab34710), rabbit polyclonal and collagen type IV (1:200, Abcam ab6586), rabbit polyclonal for 60 min followed by secondary HRP rabbit/mouse antibodies (Dako K5007) treatment for a further 30 min. The protein markers were visualised as brown after adding DAB substrate and counterstained for the nucleus with haematoxylin.

The lung tissue resections were deparaffinised using xylene and ethanol following antigen retrieval using a Decloaking Chamber (Biocare Medical, Pacheco, CA, USA) at 110°C for 15 min with target retrieval citrate buffer pH 6.0 (Dako S2369) and at 95°C for 15 min with retrieval buffer pH 9.0 (Dako S2367), respectively. The tissues were stained with EndMT primary antibodies, polyclonal rabbit anti-human S100A4 (1:1000; Dako A5114), VE-cadherin (CD144) mouse monoclonal (1:150; Thermofisher 14144982), vimentin mouse monoclonal (1:200; Dako M7020) and mouse monoclonal anti-N-cadherin (1:100; Abcam Ab98952) for 60 min; with inflammatory markers, including mouse anti-human CD4 (1:50, Dako M7310), mouse anti-human CD8 (1:50, Leica Biosystems, NCL-CD8-4B11-L-CE), CD68 (1:100, Dako M0814), mouse anti-human neutrophil elastase (1:200, Dako M0752) and mouse monoclonal mast cell chymase (CMA1, 1:100, Abcam ab2377); and with the pericyte marker platelet-derived growth factor receptor-β (PDGFR-β, 1:100, LSBio, LS-C150026); followed by secondary HRP rabbit/mouse antibodies (Dako K5007). The protein markers were visualised as brown with the addition of DAB substrate (Dako K5007). The nucleus was counterstained with haematoxylin (Australian Biostain P/L).