Evaluating Cell Viability and Protein Expression

On day 0, 1.0 × 10³ cells per well were seeded in 96-well plates. At the indicated hours, sulforhodamine B (Sigma, St Louis, MO, USA) assay was used to obtain relative estimates of viable cell number as previously described in Piro et al.^{21 (link)} Western blot analyses were carried out as described previously in Melisi et al.^{22 (link)} Briefly, cell lines were washed twice with cold phosphate-buffered saline and lysed at 4 °C into RIPA buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) plus protease inhibitor mix (Sigma-Aldrich). Each lysate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with antibodies against E-cadherin, β-catenin (Abcam, Cambridge, UK), receptor tyrosine kinase-like orphan receptor 2 (ROR2), Histone H3, glycogen synthase kinase 3α/β (GSK3α/β) and p-GSK3β-(S9) (Cell Signaling Technology, Boston, MA, USA), vimentin (Dako, Glostrup, Denmark), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and γ-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive proteins were detected using an enhanced-chemiluminescence reagent (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Images were captured by LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK).