Ab17056

Oocytes were fixed for 30–60 min at 37 °C in 100 mM HEPES (pH 7; titrated with KOH), 50 mM EGTA (pH 7; titrated with KOH), 2% formaldehyde (methanol-free) and 0.2% Triton X-100, based on previously published methods51 (link). Fixed oocytes were incubated in PBS with 0.1% Triton X-100 overnight at 4 °C. Antibody incubations were performed in PBS, 3% BSA and 0.1% Triton X-100. Primary antibodies used were mouse anti-pericentrin (30, BD Biosciences 611815; 1:750), mouse anti-γ-tubulin (GTU88, Sigma T6557; 1:3,000), rat anti-tyrosinated-α-tubulin (YOL1/34, AbD Serotec MCA78G; 1:3,000) and mouse anti-PLK1 (35–206, Abcam Ab17056; 1:1,000). Secondary antibodies used were Alexa-Fluor-488-labelled anti-mouse (Molecular Probes A11029; 1:400) and Alexa-Fluor-647-labelled anti-rat (Molecular Probes A21247; 1:400). DNA was stained with 5 mg ml⁻¹ Hoechst 33342 (Molecular Probes H3570).

The glioma cell lines U251, SNB19, U373, SHG44, and LN229 and normal human astrocytes (NHAs) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C in an incubator (Thermo Fisher Scientific, Waltham, MA, USA). High‐glucose Dulbecco's Modified Eagle's medium (DMEM; Merck KGaA, Darmstadt, Germany) with 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin‐streptomycin (Solarbio, Beijing, China) were used for cell culture. Trypsin (0.25%) (Solarbio, Beijing, China) was used to dissociate the cells. Sixteen brain glioma and paired peritumoral brain edema (PTBE) tissues were collected following tumor surgical resection performed at the First Affiliated Hospital of Zhengzhou University. Collected tissues were immediately placed into liquid nitrogen. The primary antibodies for western blotting or immunohistochemistry against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (ab8245) and PLK1 (ab17056), as well as secondary antibody (ab6789), were purchased from Abcam (Cambridge, UK).

In this study, cells were seeded into 6-well dishes with cover glasses and incubated. Then, these cells were fixed with 4% paraformaldehyde/PBS at 37 °C for 15 min and permeated with PBS containing 0.5% Triton X-100 at room temperature for 15 min. Next, the cells were blocked in a blocking buffer (PBS containing 3% bovine serum albumin; Sigma-Aldrich) at room temperature for 1 h. Afterward, the cells were incubated with anti-PLK1 antibody (#ab17056, Abcam) and anti-METTL3 antibody (#ab195352, Abcam) or a-Tubulin (#sc-5286, Santa Cruz) as the primary antibody at room temperature for 1 h. Then, the cells were incubated with Goat-anti-Rabbit Alexa 488 (#A1008, Thermo-Fisher Scientific) and Goat-anti-Mouse Alexa647 (#A21235, Thermo-Fisher Scientific) as secondary antibodies at room temperature for 30 min. Furthermore, nuclei were stained with DAPI (NucBlue™ Fixed Cell Stain, Thermo-Fisher Scientific). Finally, images were captured using a BZ-X800 microscope (Keyence, Osaka, Japan). The fluorescence intensities of PLK1, METTL3, and DAPI were measured using a BZ-X Analyzer (Keyence), and 20 cells in the mitotic phase and 20 cells in the interphase were randomly selected for comparison. In mitotic catastrophe assay, similarly, 200 cells were randomly extracted, and the number of nuclei per cell was measured.