Immunoblot analysis confirmed that the highest Axl expressing cell lines (SKHep-1, SNU-449) had suppressed E-cadherin expression and strong Vimentin expression, consistent with EMT activation (Fig

Immunoblot analysis confirmed that the highest Axl expressing cell lines (SKHep-1, SNU-449) had suppressed E-cadherin expression and strong Vimentin expression, consistent with EMT activation (Fig.?1f) but no relationship with Akt phosphorylation at the Ser473 in untreated cell lysates (Supplementary Physique?2). Axl is expressed in main and metastatic human HCC tissue samples We assessed Axl and Gas-6 expression by IHC in archival, paraffin-embedded tissue samples of resected HCC, background cirrhosis and in normal controls ( em n /em ?=?10 in each group) (Fig.?1g, h). method27 (Supplementary Methods) on CompuSyn software 1.0 (Combosyn Inc., Paramus, NJ, USA). 18FDG cell uptake assay 18F-FDG uptake studies were carried out as previously explained with modifications.28 In brief, SKHep-1 cells were seeded in 12-well plates 48?h before uptake assay and treated for 6 or 24?h with indicated doses of R428. Cells were incubated with 0.74?MBq/mL 18F-FDG (PETNET, Nottingham, UK) for 60?min. Cells were trypsinised, washed three times with PBS and lysed in RIPA buffer. The radioactivity was counted on a Packard Cobra II gamma counter (Perkin Elmer) and radioactivity was normalised to applied radioactivity and protein content, as determined by BCA assay. Measurement of soluble Axl in serum Following written, informed consent (Ethics Ref. No. 17/YH/0015) plasma samples from 40 patients with HCC were obtained before sorafenib treatment (Nexavar?, Bayer Schering Pharma). Concentrations of Axl were measured using a Polydatin (Piceid) commercial sandwich ELISA kit (EHAXL, ThermoFisher Scientific, Waltham, MA, USA) according to manufacturers instructions. Cell cycle analysis Cells were treated with R428 for 24?h then collected, fixed with ethanol and stained with propidium iodide in PBS for 3?h. Cell cycle distribution was decided using circulation cytometry (FACS Canto, Becton Dickinson, Oxford, UK) and analysed using the FlowJo software (Treestar Inc., Ashland, OR, USA). In each analysis, 10.000 events were recorded. Migration and invasion assays 50,000 cells were seeded in 300?L of serum-free media in 24-wells, 8.0?m pore transwell chambers (Corning, Corning, NY, USA). Lower chambers were filled with 10% FCS medium. Drug treatment was applied to both chambers. Following 18?h incubation, membranes were fixed in real methanol and stained with 0.4% crystal violet in 20% methanol. Non-migrated cells were removed with a cotton swab. The number of invasive cells was quantified in triplicate on 20 magnification photographs. Cell migration and invasion in response to R428 was further evaluated using real-time cell analysis (RTCA) using the xCELLigence platform (Acea Bioscience, San Diego, CA, USA) as previously explained. Cell index (CI) values at landmark timepoints were analysed across experimental conditions (Supplementary Methods).29 Wound healing assays Cells were plated in 12-well tissue culture plates and managed until 95% confluent. After overnight starvation in serum-free media, a scrape was made around the cell monolayer using a 200?L sterile micropipette tip. Initial space widths (0?h) and residual space widths at 8?h were determined from photomicrographs. Cells were subjected to transfection or drug treatment prior to plating and managed Polydatin (Piceid) in drug-conditioned media throughout the experiment. Matrigel clonogenic assay Single cell suspensions (12.500/mL) were plated on a matrigel-coated 8-well slide (Sigma Aldrich) and resuspended in full media containing 2% matrigel. Phenotypic characteristics of colonies were evaluated 7 and 14 days after treatment on 20 magnification photographs. For drug treatment with R428, media were changed every 3 days. Antibody arrays We used the Pathscan RTK Antibody Array kit (7982, Cell Signaling Technology) to simultaneously evaluate 28 RTK and 11 signalling nodes in sorafenib-naive and resistant clones. Transmission intensities were analysed using ScanAlyze array software (Eisen Polydatin (Piceid) Lab Software) and normalised transmission intensity values were derived as explained before.30 Immunohistochemistry Expression of Axl and Gas-6 was analyzed by immunohistochemistry (IHC) on primary paraffin-embedded HCC specimens following pathological review of diagnostic haematoxylin and eosin sections by a certified pathologist (F.A.M.) to identify areas of tumour and surrounding cirrhosis. Ten cases of normal liver tissue obtained from hepatectomy specimens for other indications were used as controls. The primary Rabbit Polyclonal to IL-2Rbeta (phospho-Tyr364) antibodies were incubated overnight.