Cyclin e1

For preparing cell lysates for western blotting, cells were lysed in ice-cold cell lysis buffer and centrifuged to remove cell debris. Nuclear and cytoplasmic fractions were extracted from the cell pellets as previously described [61 (link)]. After electrophoresis, proteins were transferred to a PVDF membrane. After blocking, membranes were incubated with the following primary antibodies: GAPDH (ProteinTech #60004-1-Ig), HSD17B6 (ProteinTech #11855-1-AP), AKT (CST #C6717), p-AKT (Ser473) (CST #4060), PTEN (ProteinTech #22034-1-AP), MMP2 (ProteinTech #10373-2-AP), MMP9 (ProteinTech #10375-2-AP), E-cadherin (ProteinTech #20874-1-AP), N-cadherin (ProteinTech #66219-1-Ig), Vimentin (ProteinTech #10366-1-AP), snail (CST #3895), survivin (CST #2808), GSK3β (ProteinTech #22104-1-AP), p-GSK3β (CST #9322), β-catenin (CST #8480), cyclin D1 (ProteinTech #60186-1-Ig), cyclin E1 (ProteinTech #11554-1-AP), PCNA (ProteinTech #10205-2-AP), and Lamin A/C (ProteinTech #10298-1-AP) overnight at 4 ˚C. Then, the membranes were incubated with secondary antibodies: HRP-conjugated anti‑rabbit IgG (ProteinTech #SA00001‑2) or anti‑mouse IgG (ProteinTech #SA00001‑1) for 1 h at room temperature. All the experiments were performed in triplicate.

Antibodies used in this study are as follow: CDC7, MCM2, PARP, Caspase3, cleaved-Caspase3, Bcl-2, Bax, Cyclin E1, Cyclin D1, and GAPDH were purchased from Proteintech Group, UK. Phospho-MCM2 (Ser139) was purchased from Affinity, USA. Chk1 and phospoh-Chk1(Ser 345) was purchased from CST, USA.

Western blotting was performed according to the standard protocol. The primary antibodies, including GTSE1(Proteintech, 21,319–1-AP), Ki67(Proteintech, 27,309–1-AP),mutant p53(Abcam, ab32049), p53(Proteintech, 10,442–1-AP),Cyclin D1(Proteintech, 60,186–1-Ig),Cyclin E1(Proteintech, 11,554–1-AP), p21(Proteintech,60,214–1-Ig), E-Cadherin (Proteintech, 20,874–1-AP), Desmoplakin (Proteintech,25,318–1-AP), N-Cadherin (Proteintech,22,018–1-AP), Vimentin (Proteintech, 10,366–1-AP), Nanog (Proteintech, 14,295–1-AP), ABCG2(CST, 42078S), TAZ (CST, 4883S), YAP (CST, 14074S), ERK1/2(Affinity, AF0155), phospho-ERK1/2(Affinity, AF1015), AKT (CST, 2938S), Phospho-Akt (Thr308) (CST, 13038S), Phospho-Akt (Ser473) (CST, X4060S), FoxC2(CST, 12974S),Slug (CST, 9585 T),Twist1(CST, 46702S),Snail (Proteintech, MG-3879 T) and GAPDH (Proteintech, 60,004–1-Ig) were used at a dilution of 1:1000.

Antibodies against KIFC3 were purchased from Abcam (Cambridge, MA, United States). Antibodies against proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2, MMP-9, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), p21, Cyclin A2, Cyclin E1, CDK2, E-cadherin, N-cadherin, Vimentin, Snai1 were purchased from Proteintech (Rosemont, IL, United States). Antibodies against p53, TWIST1 were obtained from Affinity (Cincinnati, United States). Antibodies against total phosphatidylinositol 3-kinase (t-PI3K), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), total AKT (t-AKT), phosphorylated Akt (p-AKT), total mammalian target of rapamycin (t-mTOR), phosphorylated mammalian target of rapamycin (p-mTOR) were obtained from Cell Signaling Technology (Danvers, MA, United States). Antibodies against PCNA, GAPDH, E-cadherin, N-cadherin, Vimentin, were used at a working concentration of 1:5000, and the other antibodies were used at a working concentration of 1:1000 and were stored at 4°C. The secondary antibodies were purchased from Proteintech (Rosemont, IL, United States) and used at a dilution ratio of 1:10,000.

During the study, various antibodies were employed, including PELI1 (ab199336, Abcam) for Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and immunoprecipitation (IP), Cyclin E1 (11554–1-AP, Proteintech), Cyclin D1 (60186–1-Ig, Proteintech), CDK7 (#2916, CST), CDK4 (GB111602–100, Servicebio), p27 (25614–1-AP, Proteintech), E-cadherin (20874–1-AP, Proteintech), N-cadherin (22018–1-AP, Proteintech), Vimentin (10366–1-AP, Proteintech), Snail (10399–1-AP, Proteintech), Alpha Tubulin (66031–1-Ig, Proteintech), RPS3 (66046–1-Ig for WB/IHC/IF, 11990–1-AP for IP, Proteintech), Ki67 (27309–1-AP, Proteintech), PCNA (10205–2-AP, Proteintech), p53 (66031–1-Ig for WB, 10442–1-AP for IP/mIHC, Proteintech), Flag (66008–4-Ig, Proteintech), HA (66006–2-Ig, Proteintech), Myc (60003–2-Ig, Proteintech). Fluorescent secondary antibodies included mouse secondary antibody (SA00009–1, Proteintech) and rabbit secondary antibody (SA00013–4, Proteintech).
Chemical reagents employed in the experiment consisted of CHX (Aladdin, China), MG132 (Sigma, USA), four-color multiplex immunohistochemistry kit (Absin, abs50012, China), PI (Solarbio, China), DAPI (Solarbio, China), and D-Luciferin Potassium salt (PerkinElmer, USA).

The following antibodies were used in the study: phospho-Rb T821 (Thermo Fisher Scientific, Waltham, MA, USA, 1:1000 dilution), Rb (Abcam, Cambridge, UK, 1:2000 dilution), phosphor-CDK2 T160 (Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution), CDK2 (Abcam, Cambridge, UK, 1:1000 dilution), Cyclin E1 (Proteintech, Wuhan, China, 1:1000 dilution), p53 (Proteintech, Wuhan, China, 1:5000 dilution), MDM2 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:1000 dilution), and β-actin (Beyotime, Shanghai, China, 1:1000 dilution).
PF-07104091 and Compound III-13 were synthesized by WuXi AppTec (Shanghai, China).

Proteins were prepared, and western blots were performed as described previously [41 (link)]. Antibodies were diluted according to the manufacturer’s instructions, including JAM3 (Abcam, USA), Caspase3/Cleaved-caspase3 (Proteintech, USA), MMP2 (Proteintech, USA), MMP9 (Proteintech, USA), cyclinD1 (Proteintech, USA), cyclin A (Proteintech, USA), cyclin E1 (Proteintech, USA), Myc (Proteintech, USA), β-catenin (Proteintech, USA), p-β-catenin (ZENBIO, China) and Actin (Proteintech, USA).