Sc 5286

Mock- or RSV-infected A549 cells were stained with MitoTrackerRed CMXRos (M7512, ThermoFisher Scientific; 100 nM, 15 min) and then fixed, washed and stained using standard protocols (Hu et al., 2013 (link)). Primary antibodies used were: anti-RSV antibody (1:400, ab20745, abcam), anti-γ-tubulin antibody (1:300, 66320–1-Ig, Proteintech), or anti-α-tubulin (1:100, sc-5286, Santa Cruz), with dye-tagged secondary antibodies (anti-goat Alexa Fluor 488, 1:1000, A-11055, or anti-mouse Alexa Fluor 647, 1:1000, A-31571, ThermoFisher Scientific) as appropriate. F-actin was stained by Alexa Fluor 488 phalloidin (1:1000, A12379, ThermoFisher Scientific). In all analyses of stained fixed cells, nuclei were stained by DAPI (1:15,000 in PBS, 10236276001, Sigma). Following mounting onto glass slides with Biomeda Gel Mount (ProSciTech), imaging was conducted using a Leica TCS SP5 channel confocal and multiphoton microscope (63X objective, oil immersion). Images (512 × 512 pixels, 8- or 12-bit) were collected and viewed using the Leica Application Suite Advanced Fluorescence Lite Version: 2.8.0 build 7266 viewer software. Airyscan super-resolution imaging was performed using the Zeiss CLSM 800 with Airyscan detector; images (2448 × 2448 pixels, 16-bit) were viewed using the ZEN 2 (blue edition) software.

Antibodies used for immunoblotting were anti-SUMO2/3 (M114-3, MBL), anti-UBC9 (sc10759, Santa Cruz Biotechnology), anti-PIAS1 (ab77231, Abcam), anti-tubulin (sc5286, Santa Cruz Biotechnology), anti-laminB1 (sc6216, Santa Cruz Biotechnology), anti-Pol2 (sc-899, Santa Cruz Biotechnology), anti-histone H3 (ab1791, Abcam), and anti-HSF1 (ADI-SPA-901; ENZO Life Sciences). Antibodies used for ChIP-seq were anti-SUMO2/3 (M114-3, MBL), anti-PIAS1 (ab77231, Abcam), and anti-Pol2 (MMS-126R, Covance).

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.

Three days post siRNA transfection, cells were lysed with lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and 1 mM DTT) and extracts were prepared after centrifugation at 13,000g, 4 °C for 10 min. Proteins were resolved in 4–15% Mini-PROTEAN TGX gels (BioRad), electroblotted to nitrocellulose membrane, and visualized using antibodies against WRN (in house), DNA-PKcs (G4; Santa Cruz), 53BP1 (C19, BD biosciences), DNA ligase IV (sc-271299, Santa cruz), Ku80 (C20; Santa Cruz), Ku70 (N3H3, Santa Cruz), CtIP (14-1, Active Motif), Artemis (N3C3, GeneTex), XLF (ab33499, Abcam), DNA ligase I (sc-20222, Santa Cruz), DNA ligase III (1F3, GeneTex), PARP1 (4C10-5, BD biosciences), XRCC1 (GTX23133, GeneTex), actin (sc-1616, Santa Cruz) and tubulin (sc-5286, Santa Cruz). Further details on the antibodies can be found in Supplementary Table 1. Uncropped images of immunoblots are shown in Supplementary figure 7.

Cells in the NVU were individually scraped down and lysed on ice for 10 min. After centrifugation (13,000g, 4°C), the resulting supernatant was saved as the cytoplasmic extract sample and the nuclear pellet was prepared for a nuclear extract sample. The samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (P0012A, Beyotime, China) and then transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h and incubated overnight at 4°C with the following antibodies: rabbit polyclonal antibody against GAP-43 (1 : 1000, 8945S, CST, China), rabbit polyclonal against AQP-4 (1 : 1000, ab31721, Abcam, China), rabbit polyclonal antibody against Claudin-5 (1 : 1000, ABT45, Millipore, China), and mouse polyclonal antibody against Tubulin (1 : 200, sc-5286, Santa Cruz, China). Membranes were incubated with a secondary goat anti-rabbit/mouse antibody (1 : 3,000, Service, China) for 1 h at 37°C. Immunoreactive bands were observed using the ECL detection system (Bio-Rad, Beijing, China).

HIF1, NOX1 and phopho-Histone H2A.X expression was analyzed in cells grown in hypoxic conditions (1% O₂ for 24 h) using specific antibodies from BD Transduction Laboratories, Santa Cruz Biotechnology (sc-5281) and Cell Signaling (#9718), respectively. To analyze the phosphorylation levels of ERK1/2 and AKT, cells were deprived of serum for 12 h before lysis and the proteins were detected using specific antibodies (Cell Signaling). Tubulin immunodetection (sc-5286, Santa Cruz Biotechnology) was used as a control of protein loading. Appropriate horseradish peroxidase coupled secondary antibodies were used and peroxidase activity was assessed by enhanced chemoluminiscence (Amersham).