Cytokeratin 18

Dulbecco’s modified Eagle medium (DMEM; #08458-45) and RPMI-1640 were obtained from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS; #172012) and hepatocyte growth factor (HGF; #H1404) were procured from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against c-Met (C-28; #161), E-cadherin (#8426), and cytokeratin-18 (#6259) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against CD9 (EXOAB-CD9A-1), CD63 (EXOAB-CD63A-1), CD81 (EXOAB-CD81A-1), and α-SMA (#14395-1-AP) were obtained from Proteintech (Rosemont, IL, USA). Antibodies against β-actin (#4967), phospho-AKT (Ser473; #9271), extracellular signal-regulated kinase (ERK) 1/2 (3A7; #9107), phospho-ERK 1/2 (p-ERK 1/2; Thr202/Tyr204, E10; #9106), and AKT (#9272) were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 3000 (#L3000008), TRIzol (#15596-018), and anti-phospho-c-Met antibody (pypypy1230/1234/1235; #44888G) were obtained from Life Technologies (Carlsbad, CA, USA). The antibody against vimentin (#M0725) was procured from DAKO (Glostrup, Denmark). Streptavidin 10 nm gold (#AC-10-04-05) was purchased from Cosmo Bio (Tokyo, Japan).

GW4064 from Tocris Bioscience (Bristol, UK). L-glutamine, penicillin, streptomycin, aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, NP-40, MTT and anti-αSMA antibody were purchased from Sigma (Milan, Italy). Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM-F12, RPMI, McCoy’s5A, Hank’s balanced salt solution, fetal bovine serum (FBS), Leptin, TRIzol, TaqDNA polymerase, RETROscript kit, 100-bp DNA ladder were from Life Technologies (Monza MB, Italy). Antibodies against FXR, Survivin, Cyclin D1, Ob, KI67, Cytokeratin 18, and β-Actin, by Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-SOCS3 antibody by Abcam (Cambridge, UK). Antibodies against total non-phosphorylated and Phosphorylated (p) JAK2 (Tyr^1007/1008), STAT3 (Tyr⁷⁰⁵), Akt (Ser⁴⁷³), and MAPK (Thr²⁰²/Tyr²⁰⁴) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cells were washed twice with cold PBS, and then lysed with RIPA lysis buffer containing protease inhibitor. After quantification, 30 μg total proteins were loaded into 10% SDS-PAGE gel and then blotted onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1 h then incubated with primary antibodies against IFIT5 (Proteintech), GAPDH (KangChen), E-cadherin, N-cadherin, vimentin, Cytokeratin-18 and ICAM1 (Santa Cruz) overnight at 4 °C. After washing with TBST for three times, the membrane was then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. After washing with TBST for another three times, the bands were visualized by ECL system (Bio-Rad Laboratories). GAPDH was used as an internal control.

Cells were fixed for 15 minutes in 4% paraformaldehyde, rinsed with PBS and permeabilised for 10 minutes in 0.1% Triton X-100. Nonspecific immunoglobulin binding was blocked with a 30 minute incubation in 3% bovine serum albumin +0.1% Triton X-100. Primary antibodies (E-cadherin Cell Signalling 24E10, Vimentin Cell Signalling 5741P, Wt1 Abcam 89901, Cytokeratin18 Santa Cruz 31700) were diluted 1:100 in 1% bovine serum albumin +0.1% Triton X-100. After overnight incubation at 4 °C, cells were rinsed with PBS, then incubated for 1 h at room temperature with secondary antibodies (1:500 Alexa Fluor Invitrogen) or Phalloidin (Invitrogen A12379). Vectashield with DAPI (Vector Laboratories) was used to counterstain the nuclei and mount the slides.

Cells were lysed in SET buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and 1% SDS) and the protein concentration was quantified by the BCA assay (#23225, Thermo Fisher Scientific). Equal amounts of whole cell extracts (ranging from 20 to 80 μg of protein depending on the antibody) were resolved in 10% SDS-PAGE gels and transferred to PVDF membranes (#ISEQ00010, Millipore). Membranes were blocked with 5% non-fat dry milk diluted in PBS-T (1x PBS, 0.1% Tween-20) for 1 h and incubated overnight at 4°C with the appropriated antibodies: HA (1:1000 dilution, #11867423001, Roche), β-tubulin (1:5000 dilution, #T4026, Sigma-Aldrich), E-Cadherin (1:1000 dilution, #610181, BD Transduction Labs), Occludin (1:1000 dilution, #71–1500, Invitrogen), PERK (1:1000 dilution, #5683 T, Cell Signaling), Cytokeratin-7 (undiluted, #790–4462, Ventana Medical Systems) and Cytokeratin-18 (1:1000 dilution, #51582, Santa Cruz). Membranes were then incubated with the corresponding secondary antibodies (rabbit anti-mouse IgG/HRP, #P0260, Dako, and goat anti-rat IgG/HRP, #A9037, Sigma-Aldrich) at a 1:5000 dilution. Membranes were visualized using chemiluminescence reagent (#WBLUF0500, Millipore) and exposed on Odyssey Fc Imaging System (Li-Cor).