Cyclind3

Equal amounts of protein were solubilized in sample buffer and electrophoresed in denaturing 10% SDS-polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were saturated in TBST containing 5% BSA (Bio Sharp Sigma, A-4612) for an hour at room temperature and incubated with primary antibodies overnight at 4 °C. After incubation with a secondary antibody, the blots were then washed and detected with the ChemiDoc MP System (Bio-Rad Laboratories. Inc., Hercules, CA, USA). The antibodies used are as follows: GLI1 (Cell Signaling, 3538), p-AKT (Ser473) (Cell Signaling, 4060), AKT (Cell Signaling, 9272), p-PI3K (Cell Signaling, 17366), PI3K (Cell Signaling, 4257), GSK3α/β (Bioworld, BS1412), Cyclin D1 (Cell Signaling, 2978), CyclinD2 (Proteintech,10937-1-AP), CyclinD3 (Proteintech, 26755-1-AP), CDK4 (Proteintech, 11026-AP), CDK6 (Proteintech,19117-1-AP), GAPDH (Santa Cruz, CA, USA), and anti-rabbit IgG (Proteintech, SA00001-2).

Western blotting was performed according to the procedures described previously [20 (link)], loading 25 μg of total protein lysate per lane. Primary antibodies were diluted at 1:1000 and secondary antibodies were diluted at 1:5000. Antibodies against the following proteins were used for the study: HDAC7 (#33418), USP10 (#8501), β-catenin (#8480), acetyl-β-catenin (Lys49; #9030), phospho-β-catenin (Thr41/Ser45; #9565), phospho-β-catenin (Ser552; #5651), phospho-β-catenin (Ser675; #4176), Cyclin E1 (#4129), E-cadherin (#14472), Slug (#9585), β-actin (#3700), Lamin B1 (#13435), and GAPDH (#5174) (CST). HDAC7(26207–1-AP), CDK1(19532–1-AP), CDK2 (10122–1-AP), CDK4 (11026–1-AP), CDK6 (14052–1-AP), Cyclin A2 (66391–1-Ig), Cyclin B1 (55004–1-AP), Cyclin D1 (60186–1-Ig), Cyclin D3 (26755–1-AP), FGF18 (60341–1-Ig), FGFR3 (66954–1-Ig), Snail (13099–1-AP), vimentin (10366–1-AP), ZEB1 (21544–1-AP), Flag (66008–3-Ig), and ubiquitin (10201–2-AP) were also used (Proteintech Group).

Bortezomib was purchased from LC Laboratories (Woburn, MA, USA). For in vitro studies, the drug was reconstituted in dimethyl sulfoxide (DMSO) at a stock concentration of 1 mM and stored at −80°C. The small‐molecule inhibitors MKC3946 and GSK2606414 were bought from MedChemExpress (Monmouth Junction, NJ, USA). The stock solution for MKC3946 and GSK2606414 was prepared in DMSO at a concentration of 10 mM and stored at −20°C. The antibodies against p21^Cip1, p27^Kip1, X‐box‐binding protein 1 (Xbp1s), activating transcription factor 6 (Atf6), 78 kDa glucose‐regulated protein (Grp78), C/EBP homologous protein (Chop), cyclin D3, cyclin E1, cyclin‐dependent kinase 2 (CDK2), cyclin‐dependent kinase 4 (CDK4) and β‐actin were obtained from Proteintech (Wuhan, Hubei, China), and the antibody against activating transcription factor 4 (Atf4) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). All other chemicals were obtained from Sigma‐Aldrich (Burlington, MA, USA) unless otherwise specified.