Calyculin a

Protein phosphatase 1 and 2A inhibitor (Calyculin A), protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ22,536) were purchased from Sigma-Aldrich. The following primary antibodies were used for western blotting: myosin light chain 2 antibody (#3672; Cell Signaling Technology, MA, USA), phospho-myosin light chain 2 (Ser19) antibody (#3671; Cell Signaling Technology), phospho-myosin light chain 2 (Thr18/Ser19) antibody (#3674; Cell Signaling Technology), myosin light chain 2 antibody (#3672; Cell Signaling Technology), GAPDH antibody (#AM4300; Ambion, TX, USA), MYPT1 antibody (#2634; Cell Signaling Technology), phospho-MYPT1 (Thr696) antibody (#5163; Cell Signaling Technology), phospho-MYPT1 (Thr853) antibody (#4563; Cell Signaling Technology), RhoA antibody (#ARH03; Cytoskeleton, CO, USA), and phospho-RhoA (Ser188) antibody (#AB41435; Abcam, Cambridge, UK). MRLC stained by phospho-myosin light chain 2 (Ser19) antibody is denoted as 1P-MRLC, and MRLC stained by phospho-myosin light chain 2 (Thr18/Ser19) antibody is denoted as 2P-MRLC.

CAs were studied in MCF-10A cells at 36 h post irradiation by chemical induction of premature chromosome condensation (PCC). PCC was obtained following 30-min incubation in calyculin A (50 ng/ml, Sigma Aldrich) and collected by standard cytogenetic protocol as elsewhere described (35 (link), 36 (link)), slightly modified for adherent cells. Detection of structural CAs was carried out by Fluorescence-in Situ Hybridization (FISH) techniques: Whole Chromosome Painting (WCP) and multicolor (m)-FISH (14 (link), 37 (link)). For WCP, two pairs of homologous chromosomes were labelled with probes (MetaSystems, Germany) directed to chromosomes 1 and 2 emitting in the green (chromosome #1, XCP-1 FITC-conjugated probe) or red (chromosome # 2, XCP-2 orange) spectrum under UV light. Denaturation (72°C for 3 min) followed by hybridization (37°C for 4 h) of 72-h room-temperature aged slides was performed using the programmable HyBrite chamber system (Vysis, USA). After post-hybridization washes, chromosomes were counterstained by DAPI/antifade (250 ng/ml). For mFISH, the 24XCyte probe cocktail, made up of five fluorochromes by MetaSystems (CyTM5, DEAC, FITC, Spectrum OrangeTM, Texas Red), was applied to PCC spreads harvested as described above. A detailed protocol can be found in Cirrone et al. (14 (link)).

Embryos were mounted in agarose as described above, then embryo medium containing 50 nM Calyculin A (Sigma-Aldrich) was added approximately 30 min prior to the start of imaging and remained throughout the imaging period.

To inhibit ROCK, cells were pre-treated for 20 min with Y27362 (Cat#Y0503, Sigma-Aldrich) at a final concentration of 10 μM. For suspension analysis, following detachment with Accutase (Cat#A1110501, ThermoFisher Scientific) for 5 min, cells were collected into complete DMEM (+/− Y27362), centrifuged at 500 xg for 5 min and resuspended in complete DMEM (+/− Y27362). To increase cell contractility, Calyculin A (Cat#C5552, Sigma-Aldrich) at final concentration of 25 nM was used and live imaging started immediately following its addition. Ezrin was inhibited with NSC668394 (Cat#341216, Merck Millipore) overnight at a final concentration of 10 μM or for 3 hr at 250 μM. For actin depolymerisation Latrunculin B (Cat#L5288, Sigma-Aldrich) was used at a final concentration of 20 nM. Equivalent volume of DMSO was used as controls. Accutase treatment and following incubation in suspension were performed at room temperature (22°C–25°C, RT). Accutase treatment had no effect on cell viability or ability to respread (Figure S1). Cells were also detached using EDTA (0.68M, in-house prepared solution).

Cell culture was performed as reported previously^{23 (link)} and reiterated here. COS-7 cells were obtained from ATCC (Manassas, VA, USA, catalog number CRL-1651) and maintained in standard DMEM-HG medium supplemented with 10% (vol/vol) heat-inactivated FBS, 1 mM sodium pyruvate and 2 mM Glutamax in a 5% CO₂ incubator at 37 ^°C. For imaging, the cells were plated in Bioptechs Delta-T dishes (Bioptechs, Butler, PA, USA, product number 04200417B) and grown to ~ 70% confluency. Transient transfections were done with various plasmids according to manufacturer’s instructions using 1–2 μg DNA and 3ul of X-tremeGene HP DNA transfection reagent (Roche, Manheim, Germany, product number 06366236001). The transfected cells were incubated at 37 °C and 5% CO₂ incubator for 24–48 h before imaging. The cell culture reagents DMEM-HG medium (catalog number 11960), fetal bovine serum (FBS, catalog number 10082), sodium pyruvate (catalog number 11360) and Glutamax (catalog number 35050) were obtained from Invitrogen (Life Technologies, Grand Island, NY, USA). Calyculin A was obtained from Sigma (catalog number C 5552).