Aphidicolin

MEFs were seeded at 40,000 cells/ml on Ibidi 8-well µSlides (IB-80826, Ibidi) and allowed to attach for 48 h before treatment. All cells were treated with either mitomycin C (MMC) or aphidicolin (APH) at day 6 post recombination. For MMC assays cells were treated with 2 µM of MMC (Sigma) for 2 h before replacing with either fresh media or media containing 200 nM of CDKi (CDK1/2 Inhibitor III, 217714, Merck Chemicals) as specified. Alternatively, cells were treated with 75 nM of Chaetocin for 24 h before adding 2 µM of MMC for 2 h. After treatment, fresh media containing 75 nM of Chaetocin was added for a further 24 h. For APH assays, aphidicolin (A4487, Sigma) was added at 200 nM with or without 200 nM of CDKi for 24 h. Alternatively, cells were treated with 75 nM of Chaetocin for 24 h before adding aphidicolin 200 nM to the media for a further 24 h.

Synchronization was performed as previously described^{19 (link)}. Briefly, cells were plated in INS-1 RMPI media containing 10% FBS. Asynchronous cells were plated at the same time, but remained in INS-1 media for the remainder of the study. After 24 h, synchronized cells were switched to INS-1 media + 0.1% FBS for 56 h, at which point cells were treated with 2 µg/ml aphidicolin (Sigma-Aldrich) for an additional 12 h. Following aphidicolin treatment, media was changed to INS-1 media with 10% FBS and cells were collected at 0 h, 4 h, and 12 h for flow cytometry analysis of cell cycle phases and RNA content. To quantify miRNA export, media was changed to serum free media or serum free +1 mg/mL nHDL 4 h prior to the final time-point for each condition. For analysis of cell cycle phases by flow cytometry, cells were trypsinized and counted, and 5 × 10⁵ cells were washed with 1X PBS and fixed in 80% methanol (Sigma-Aldrich) at −20 °C overnight. methanol was removed by centrifugation at 2000 × g for 10 min and cells were washed in 1X PBS twice. Cells were stained with 50 μg/ml propidium iodide (Invitrogen) in the presence of 50 μg/ml RNAse A (ThermoFisher) on ice for 2 h. DNA content was analyzed using the 3-laser BD LSRII at the Vanderbilt Flow Cytometry Shared Resource Core.

For NCS treatment, medium was replaced with fresh medium supplemented with neocarzinostatin (N9162, Sigma‐Aldrich) during experiments. For myc overexpression, RPE cells were infected with fresh retrovirus containing MSCV‐Myc‐ER‐IRES‐GFP and 1 μl polybrene. Cells were subsequently passaged 48 of post‐infection and seeded onto a glass‐bottom plate for imaging. 16 h prior to imaging, tamoxifen was added at a final concentration of 50 nM. For aphidicolin treatment, medium was replaced with fresh medium supplemented with aphidicolin (A0781, Sigma‐Aldrich) for 8 h during experiments, washed off once with PBS, and then replenished with imaging media described below. For CDK2 inhibition, cells were treated with 2 μM CVT‐313 (221445, Santa Cruz) prior to starting the imaging.

H1299+shBRCA2^DOX cells were seeded in order to reach 70-80% confluence at the end of the experiment (1.5-2 million cells per T175 flask, 10-25 flasks per condition) and DOX (Sigma-Aldrich) was added where indicated. Cells were synchronized at the G1/S transition with 1.5 mM thymidine (Sigma-Aldrich) for 16 hours. To detect MiDAS under untreated conditions, cells were washed three times with PBS and released in fresh medium containing 6 μM RO-3306 (Sigma-Aldrich) for 10.5 hours. To detect aphidicolin-induced MiDAS, cells were washed three times with PBS and released in fresh medium containing 6 μM RO-3306 (Sigma-Aldrich) and 0.2 μM aphidicolin (Sigma-Aldrich) for 17.5 hours. For both protocols, cells were then washed three times with warm medium and released in medium containing 100 ng/mL nocodazole (Sigma-Aldrich) and 10 μM EdU (ThermoFisher). To suppress replication in S-phase cells that could potentially contaminate the mitotic shake-off 2 mM HU (Sigma-Aldrich) was added during the final 3 hours. Mitotic cells were collected by mitotic shake-off, fixed with 90% ice-cold methanol and stored at -20°C until processed for isolation of EdU-labelled DNA.

HeLa cells were cultured in Advanced DMEM (Life Technologies Ltd, Paisley, UK) supplemented with 2% foetal bovine serum, glutamax-I (200 µM), penicillin (100 U/ml), streptomycin (100 µg/ml) and fungizone (250 ng/ml) at 37°C, 10% CO₂. MEFs were cultured in the medium previously described (García-Higuera et al., 2008 (link)). HeLa cells were synchronised at G1/S transition by a thymidine/aphidicolin block: the day after seeding, cells were blocked with thymidine (2.5 mM, Sigma–Aldrich, Gillingham, UK) for 24 hours, released for 12 hours and then blocked again with aphidicolin (2.5 µg/ml, Sigma–Aldrich) for 24 hours. Cells were then released in fresh DMEM.