M1404

For immunoprecipitation, MCF7 cells and 293T cells were treated with thymidine (2 mm, #T1895; Sigma‐Aldrich, St. Louis, MO, USA) for 18 h and released into fresh medium for 10 h and then blocked with 2 mm thymidine again for 18 h. After that, the cells were released into fresh medium for 2 h and treated with nocodazole (0.1 μg·mL⁻¹, #M1404; Sigma‐Aldrich) for 10 h. Then the mitotic cells were collected for immunoprecipitation.
For analysis of flow cytometry and western blot, SUM159PT cells, MCF7 cells, and 293T cells were treated with nocodazole (0.1 μg·mL⁻¹, #M1404; Sigma‐Aldrich) for 10 h and released into nocodazole‐free medium; then the cells were collected at the indicated time for further analysis.

HeLa and HEK293T cells were maintained in DMEM (Hyclone) medium supplemented with 10% fetal bovine serum (Hyclone) and 1% pen-strep (Gibco 15140-122). The CHK2 knockout (KO) HeLa cell line was generated as described previously [17 (link)]. The CHK2 KO cells were transfected with constructs expressing myc-CHK2 (WT or Y156F)-FLAG to establish G418-resistant stable cell lines. These cells were regularly maintained in a medium containing 400 μg/mL G418. The normal human fibroblasts MRC5 and WI38 were maintained in MEM (Gibco) supplemented with 10% fetal bovine serum and 1% pen-strep. All cell lines were from ATCC.
For ionizing radiation treatment, cells were exposed to 8 Gy X-ray using the Torrex 150D inspection system (EG&G) and then incubated for 2 h. Where indicated, cells were treated with CHK2 inhibitor II (C3742, Sigma Aldrich) and JAK2 inhibitor IV (#420139, Calbiochem) at concentrations of 5 and 2.5 μM, respectively. For nocodazole treatment of HeLa and HEK293T (M1404, Sigma Aldrich), either 50 ng/ml (for protein analysis) or 25 ng/ml (for confocal microscopy) was used. For the mitotic arrest of normal fibroblasts WI38 and MRC5, 600 ng/ml of nocodazole was used for protein analysis and 500 n/ml was used for confocal microscopy. Turbofect (Thermo Scientific) and calcium phosphate method were used for transfection of HeLa and HEK293T cells, respectively.

The mitochondria in living cells were transfected for 24 h with Ad-Mito-EGFP, which contained the mitochondria-specific targeting sequence 5′-ATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGA-3′. Then, cocultured CMs and MFs were treated with microtubule depolymerisation reagents nocodazole (30 μM, 4 h; M1404, Sigma, St. Louis, MO, USA) or colcemid (4 μM, 4 h; 10295892001, Sigma), followed by mitochondrial tracking.
Mitochondrial movement in MNTs between CMs and MFs was tracked in 30 s intervals for 15–20 min using a confocal fluorescence microscope with a 63X/1.4NA oil immersion objective lens (Zeiss Laser Scanning Microscope, LSM780, Zeiss, Oberkochen, Germany). A cell incubation system (Incubator PM S1) was used to ensure that the cells were imaged under 37 °C and 5% CO₂ conditions. For living cells, we were able to distinguish between CMs and MFs because only CMs can beat. AxioVision 4 Tracking Module (Zeiss) and ImageJ (NIH, Bethesda, MA, USA) were used to track and analyse mitochondrial movement velocity in MNTs.

Jurkat cells were washed, resuspended at 1 × 10⁶ cells/mL and incubated 4 h with nocodazol (5 μg/mL, M1404, Sigma-Aldrich, St. Louis, MO, USA) in RPMI containing 10% of FCS at 37 °C. 150,000 Jurkat cells were then incubated on coverslip for 30 min, washed once with cold PBS and fixed.

Rhodamine-labelled tubulin was added to the XEE to visualise the nucleation of microtubules. Centrioles of the bipolar spindles were detected by adding Nocodazole (0.5 uM, 20 min at 20 °C, M1404 Sigma) to the XEE before spinning them down^{11 (link)}. Briefly, right after the pelleting of the samples, they were blocked with 5% BSA 0.1% Triton X-100 in PBS for 50 min at room temperature, and the monoclonal mouse anti-centrin antibody (20H5, Millipore, 1:2,000) was then used to immunodetect the centrosomes. The percentage of detected centrioles at both poles, one pole and no centrioles was quantified for Xenopus, human normozoospermic and asthenozoospermic sperm samples in four independent experiments.