Ccne1

Total proteins were prepared from the samples by complete cell lysis (Keygen Biotech, Jiangsu, China) with protease and phosphatase inhibitors. Identical quantities of proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene difluoride membranes. After incubation with antibodies specific for HBx (Abcam, Cambridge, UK), CDK6 (Cell Signaling Technology, Beverly, MA, USA), CCND1(Cell Signaling Technology), CCNE1(Cell Signaling Technology), p16(Cell Signaling Technology), p21(Cell Signaling Technology), p27(Cell Signaling Technology), Caspase3(Immunoway, USA), cleaved Caspase3(Cell Signaling Technology), ERK (Cell Signaling Technology), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), JNK(Cell Signaling Technology), p-JNK(Cell Signaling Technology), p53(Millipore, Schwalbach/Ts., Germany), HIF-1α(Abcam), GAPDH (Cell Signaling Technology) and β-actin (Proteintech, USA), the blots were incubated with goat anti-rabbit or anti-mouse secondary antibodies (Bioss, Beijing, China). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blotting Detection System; Amersham Biosciences, Foster City, CA, USA) and quantified by ImageJ software.

Protein was extracted from GAC cell lines and paired primary tissues using RIPA lysis buffer with proteinase inhibitor. Protein concentration was measured by the method of Bradford (Bio-Rad, Hercules, CA) and 20 μg of protein mixed with 2 × SDS loading buffer was loaded per lane, separated by 12% SDS-polyacrylamide gel electrophoresis. Horseradish peroxidase (HRP) substrate solution was used for signal detection (Millipore, Billerica, MA). YAP1 protein was detected with a monoclonal anti-YAP1 antibody (1:10000 dilution, ab52771, Abcam, Cambridge, MA). Other primary antibodies were obtained from Cell Signaling Technology (Danvers, MA), CCND3 (1:2000, #2936), CCNE1 (1:1000, #4129), CDK6 (1:2000, #3136), p21 (1:1000, #2946), p27 (1:1000, #2552), p-Rb(Ser807/811) (1:1000, #9308), cleaved PARP(Asp214) (1:1000, #9541), BCL2(1:1000, #2870). The secondary antibodies were anti-Mouse IgG-HRP (1:30000 dilution, 00049039, Dako, Glostrup, Denmark) and anti-Rabbit IgG-HRP (1:10000, 00028856, Dako). The Western blot bands were quantified by ImageJ.

Western blot was carried out as previously described [17] . Primary antibodies were as follows: SNAI1 (1∶200, RnD systems), SP1 (1∶1000, Cell Signalling), CDKN1a (1∶1000, Cell Signalling), CCNE1 (1∶1000, Cell Signalling), MMP2 (1∶1000, Cell Signalling), MMP9 (1∶1000, Cell Signalling), PLAU (1∶150, Abcam), GAPDH (1∶5000, Bioworld) and β-actin (1∶5000, Bioworld).

The above cell lines (TOV-21G, KOC7c, RMG-I and ES2) were grown in six-well dish (4.0 × 10⁵ cells per well) and si-CCNE1 and si-control were reverse transfected at 5 or 10 nM according to the manufacturer’s recommended protocol. Then, we extracted protein at 48 and 72 h after transfection. Samples were applied to Mini-PROTEAN® TGX^TM Gels 4–15% and transferred by Trans-Blot® Turbo^TM Transfer Pack (BIO-RAD, Hercules, CA, USA). The following antibodies were used for western blotting: primary antibodies against CCNE1 (#20808, Cell Signaling TECHNOLOGY, San Diego, CA, USA; diluted 1:2000), ARID1A (#12354, Cell Signaling TECHNOLOGY, San Diego, CA, USA; diluted 1:2000) and ß-actin (#4970, Cell Signaling TECHNOLOGY, San Diego, CA, USA; diluted 1:10,000). Horseradish peroxidase-conjugated secondary antibodies against rabbit (sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:10,000) were used.

The protocols for western blot were well described previously 34. The primary antibodies against p21, pRb, Rb, E2F1, CCNE1 and CCND1 (Cell Signaling Technology, Boston), and β‐actin (Santa Cruz Biotechnology, Dallas) were used in this study.