Nocodazole

Calcium Green-1/AM and Fura Red/AM were obtained from Invitrogen (MA, United States). Nocodazole was obtained from Tocris (United Kingdom). The pNaKtide peptide was obtained from HD Biosciences (China) and stock solution was prepared in water (10 mM) and stored at −20°C. All other chemicals were purchased from Sigma-Aldrich (Denmark). Stock solutions of Nocodazole, genistein and PP2 were prepared in DMSO (10 mM) and stored at −20°C. Ouabain stock solution was prepared on the day of experiment (minimum 2 h prior to application) in a concentration of mM in water. Drugs were applied a minimum 15 min prior to measurements/interventions.

For the alternate MiDAS protocol, a different synchronization strategy was used in order to study DNA synthesis in cells that had already entered mitosis. Asynchronously growing cells, at 70% confluency, were treated for 8 h with 100 ng/mL nocodazole (Tocris, Cat. No. 1228) and 0.4 µM aphidicolin (Sigma-Aldrich, A0781) to induce DNA replication stress in S phase cells before arresting them in prometaphase. Negative control cells were obtained by treating the cells with nocodazole but omitting aphidicolin. After the 8 h treatment, mitotic cells were shaken-off, washed with PBS and released for 4 h into warm medium with 2 mM hydroxyurea (Sigma, Cat. No. H8627) and 25 µM EdU (Invitrogen, Cat. No. A10044) to label DNA synthesis from prometaphase to early G1 phase. The cells were finally collected by trypsinization, fixed with 90% methanol overnight and EdU-labeled DNA was isolated and sequenced as described in the main MiDAS protocol.

CRC cell lines HCT116, HCT-15, and RKO, and normal colon epithelial cells CCD 841 CoN were acquired from the American Type Culture Collection. CRC cell lines were cultured in phenol red–free RPMI 1640 (PAN-Biotech), and CCD 841 CoN, in DMEM (PAN-Biotech), each supplemented with 10% charcoal-stripped fetal calf serum (Th. Geyer GmbH) and 1% penicillin/streptomycin (Merck Millipore) at 37°C and 95% humidity and 5% CO₂. Culture medium conditions were used for all experiments. All chemicals were purchased from Sigma-Aldrich unless otherwise stated, dissolved in DMSO, and stored at −20°C. The cells were treated with 10 nM E2, 10 nM BPA, 10 nM DES, 5 nM nocodazole (Noc), 10 nM tamoxifen (Tam), 100 nM G15 (Tocris Bioscience), 100 nM G36 (Biomol), 100 nM ICI182,780 (ICI), and 100 nM G-1 (Tocris Bioscience) for 48 h in a stripped medium unless otherwise stated.

For inhibition of TRPA1, we incubated cells with the antagonist HC-030031^{13 (link)} (40μM in DMSO; Cayman Chemicals #11923) for 45 min before stimulation. For activation of TRPA1, we used NMM^{13 (link)} (N-Methylmaleimide; 100μM in DMSO Sigma-Aldrich #389412) or AITC^{28 (link)} (30μM in DMSO, allyl isothiocyanate; Sigma-Aldrich # 377430). For activation of Piezo1, we used yoda-1⁷⁵ (10 μM in DMSO; Tocris #5586). For activation of TRPV1 we used capsaicin^{76 (link)} (3 μM in DMSO; Sigma-Aldrich #M2028). The final concentration of DMSO in the external solution was 0.1% or lower for all groups, which was also used as vehicle control. For cytoskeleton experiments, nocodazole (5μM; Tocris, #1228), jasplakinolide (200 μM; ThermoFisher # J7473), paclitaxel (600 nM; Sigma-Aldrich # T7191), cytochalasin D (5μM; Cayman Chemicals, #11330) or latrunculin A (1 μM; Cayman Chemicals, # 10630) in 0.1% DMSO were added to the culture media 45 min prior to imaging^{37 (link)}. For pharmacology in primary neurons, we used TRPV1 antagonist, A784168^{77 (link)} (20μM; Tocris, #4319, 45 min incubation) in 0.1% DMSO, BAPTA^{78 (link)} (30μM; Invitrogen, #B1204, 45 min incubation) directly dissolved in culture media and TTX^{79 (link)} (18μM; tetrodotoxin citrate; Tocris #1069, 5 min incubation, where we also inhibit TTX-R channels).

For synchronization of cells at the restriction point in G1, cells were washed in PBS and then media was replaced with DMEM with 0.25–0.5% FBS. After 2–3 days cells were washed in PBS, and the media containing 10% serum was added to cells. Synchronization of cells at the G2/M checkpoint was performed by treating cells with 100 ng/mL nocodazole (Sigma) in complete media overnight. The following day, cells were washed in PBS and complete media was added. Cell synchronization was assessed by flow cytometry. nocodazole and paclitaxel (Sigma) treatment of cells in G1 was performed after synchronizing cells by serum starvation for 2–3 days, and then 10–25 uM paclitaxel (Sigma), or paclitaxel and 30 ug/mL cycloheximide (Sigma) were added for 6–8 hours. Thirty uM Tame-HCl (Tocris) was added to MLE15 cells simultaneously with nocodazole to inhibit Anaphase Promoting Complex (APC) activity.