Anti wee1

Following protein harvest using Laemmli lysis buffer, protein concentrations of whole cell extracts were measured using DC protein Assay (Bio-Rad). Next, equal amounts of protein were loaded onto a Bio-Rad Mini-Protean^® TGX™ 4-20% gel. Proteins were subsequently transferred onto polyvinylidene difluoride (PVDF) membranes, after which membranes were blocked using ECL advance blocking agent (GE Healthcare) in TBS-Tween 0.1%. Proteins of interest were detected using the following antibodies: anti-N-myc (Cell Signaling, Cat: 9405), anti-CHK1 (Cell Signaling, Cat: 2360), anti-phospho-CHK1 S296 (Cell Signaling, Cat: 2349), anti-WEE1 (Cell Signaling, Cat: 13084), anti-CDC2 (Cell Signaling, Cat: 9116), anti-phospho-CDC2 Y15 (Cell Signaling, Cat: 4539), anti-γH2AX (Abcam, Cat: ab26350), anti-alpha-tubulin (Cell Signaling, Cat: 3873), anti-beta-actin (Cell Signaling, Cat: 4967). Following treatment with HRP-link secondary antibodies (Invitrogen), detection was performed using Bio-Rad Chemidoc™ Touch (BioRad).

Cell lysates were prepared with RadioImmuno Precipitation Assay buffer (Upstate). Lysates containing equal amounts of protein were separated by SDS-PAGE and electroblotted onto Immobilon™-P Transfer Membrane (Millipore). The filters were individually probed with specific primary antibody. Protein bands were detected by the Western Lightning Chemiluminescence Reagent Plus Kit (Perkin-Elmer Life Sciences) with horseradish peroxide labeled secondary antibody as suggested by the manufacturer and visualized on a VersaDoc Image System (Bio-Rad). The intensity of bands was quantified by densitometry and normalized to ACTB in each lane. Anti-human EMP2 (1:100, HPA014711, Sigma-Aldrich), anti-cyclin dependent kinase 1 (CDK1; 1:200, sc-54, Santa Cruz), anti-pCDK1(Y15) (1:250, BD Transduction Laboratories™), anti-WEE1 (1:1000, #4936, Cell Signaling), anti-CCNB1 (1:500, sc-245, Santa Cruz), anti-pCDC25C(S216) (1:1000, #4901, Cell Signaling) and anti-pCREB1(S133) (1:200, sc-7978, Santa Cruz) were used as primary antibodies for immunoblotting analysis and anti-ACTB (1:3000, Chemicon) was served as a loading control.

Cisplatin (MedChem Express, USA) was dissolved in H₂O to make a stock solution at the concentration of 1 mg/ml (3.33 mM) and stored in single-use tubes at −80°C. Anti-WTAP, anti-CDK1, and anti-BAX antibodies were supplied by Abcam (USA). Anti-Mcl-1, anti-Wee1, anti-β-catenin, anti-GSK-3β, anti-GSK-3β pSer9, anti-H2A, and anti-GAPDH antibodies were provided by Cell Signaling Technology Inc. (China). Anti-PARP antibody, anti-ki67 antibody, anti-β-actin antibody, and goat anti-rabbit IgG were obtained from ProteinTech Group Inc. (USA).

9human colorectal cancer cell lines (HCT116, LoVo, RKO, Caco2, HT29, SW48, SW480, SW620, and DLD1) used in this study were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). The human normal colonic epithelial cell line FHC was purchased from the Wuhan University Type Culture Collection (Wuhan, China). HCT116, LoVo, and SW620 cells were cultured in McCoy's 5A, F12k, and L15 media, respectively, each containing 10% fetal bovine serum (FBS).RKO, Caco2, HT29, SW48, and SW480 cells were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS. DLD1 and FHC cells were incubated in Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% FBS. All cell lines were incubated at 37 °C in a CO₂ incubator, except for SW620 cells, which were incubated exposed to air.AZD5153、BMN673 and MK1775 were bought from Selleck (Shanghai, China). The anti-c-Myc, anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-cleaved-caspase-3, anti-Wee1,anti-CDK1,anti-phospho-CDK1 (P-CDK1), anti-γ-H2AXand anti-Ki-67 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-BRD4 antibody was from Abcam (Cambridge, MA, USA), and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from Proteintech Group, Inc. (Wuhan, Hubei, People's Republic of China).