Nocodazole

Cells were treated with DMSO control or drugs diluted in DMSO at the following concentrations: cells were treated with latrunculin B (0.5, 2 μM) or cytochalasin D (0.5, 2 μM; Sigma) for 40 min prior to hydrostatic pressure experiments. Nocodazole (3 μM; abcam) treatment was carried out for 10 min. For cytochalasin D and Nocodazole combination treatment, cells were treated with cytochalasin D for a total length of 40 min while Nocodazole for a total length of 10 min at the aforementioned concentrations. For Torin and Rapamycin treatments, cells were treated at 1 and 0.5 μM, respectively, for a total of 40 min.

The CAMs were synchronized in G₂/M phase by treating the cell with nocodazole (100 ng/ml, Abcam) for 12 h [18 (link)]. After soft wash with cold PBS, the CD38^+/+ or CD38^−/− CAMs was incubated with vehicle (Vehl) or baf for 24 h and then collected by trypsinization. Furthermore, the CAMs was harvested by centrifugation on 700 rpm at 4°C for 10 min and washed twice with PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4℃ and washed twice with PBS. The cells were treated with ribonuclease (100 μg/ml, Abcam) followed by addition of 200 μL propidium iodide (PI, 50 μg/ml, Abcam). Samples were analyzed using a Guava Easycyte Mini Flow Cytometry System and the Guava acquisition and analysis software (Guava Technologies, Hayward, CA). The cell number in G0/G1, S or G2/M phase was divided by the total cell number (G0/G1+S+G2/M) to indicate the percentage of cells in specific phases.

The cell cycle synchronization protocol was adapted from a previous publication [36 (link)]. In brief, PIGA intr5_1 gRNA positive H1-iCas9 ESCs were seeded at a density of 2 × 10⁵ cells per well in a 12-well plate. To synchronize the cells at the G2/M phase, a 16-h treatment with 100 ng/ml nocodazole (Abcam, Cat# ab120630) was administered. Subsequently, the cells were washed twice with prewarmed 1 × PBS and then cultured in fresh E8 medium for 4 h or 12 h to release cells in the G1 or S phase, respectively. Alternatively, the synchronized cells were treated with 2 µg/ml doxycycline to induce Cas9 expression and genome editing, followed by 10 days’ culture for LD analysis.
Cell cycle analysis was performed using a standard protocol. Initially, the cell pellets were fixed by adding cold 70% ethanol dropwise while vortexing and then incubated overnight at − 20°C. Subsequently, the cells were washed twice and resuspended in FACS buffer containing 200 µg/ml rNase. After a 20-min incubation at room temperature, the cells were washed once and resuspended in FACS buffer containing 1 µg/ml propidium iodide (PI). Following a 10-min incubation at room temperature, the samples were ready for FACS analysis.

Thapsigargin was obtained from Cayman Chemical (Ann Arbor, MI, United States). Colcemid was purchased from Merck (Darmstadt, Germany). Tubastatin A was obtained from BioVision (Milpitas, CA, United States). Nocodazole was purchased from Abcam (Cambridge, United Kingdom). Fura-2/AM was obtained from Invitrogen. EGF Recombinant Human Protein was purchased from Thermo Fisher (Thermo Fisher Scientific, Waltham, MA, United States). The antibodies used in this study are shown in Supplementary Table 3.

To soften tumor cells, HCC cells were pretreated with different doses of Y-27632 (2 and 20 μM; Selleckchem), blebbistatin (6 and 50 μM; Selleckchem), and cytochalasin D (0.1 and 1 μM; Selleckchem) for 48 h. To stiffen them, the cells were pretreated with 20 nM jasplakinolide (Tocris, #2792/100U) or 1 nM narciclasine (MedChemExpress) for 12 h. Nocodazole (1 and 6 μM; Abcam) and withaferin A (WFA; Abcam; 1, 2, and 5 μM) were used to disrupt microtubules and vimentin, respectively. The Wnt activator LiCl (2 μM) and inhibitor IWR-1-endo (0.2 μM) were used to activate and inhibit the Wnt/β-catenin pathway, respectively.