Blebbistatin

HN30 and HN31 cells were growth arrested in serum-free medium for 24 hrs prior to drug treatment. Cells were pretreated with DMSO or 25 μM blebbistatin (Cayman Chemicals 674289-55-5) for 30 min prior to 8 hrs of treatment with 5 μM of nutlin-3 (Sigma-Aldrich N6287). For some Western blot analyses, (where indicated), 20 nM of leptomycin (Cayman Chemical 87081-35-4) was added 30 min prior to pretreatment of cells with DMSO or blebbistatin.

Chemicals were obtained from Sigma-Aldrich (Poole, UK) unless otherwise stated. Immediately following sacrifice, the heart was excised and transferred into ice-cold Krebs–Henseleit (KH) solution: (mM) NaCl, 108; NaHCO₃, 25; KCl, 4.0; KH₂PO_4, 1.2; MgCl_2, 1.0; CaCl_2, 1.8; glucose, 10; and Na-pyruvate, 2.0; pH adjusted to 7.4 and bubbled with 95% O₂/5% CO₂ (British Oxygen Company, Manchester, UK). The aorta was cannulated with a trimmed and filed 21G hypodermic needle, and secured onto the cannula with an aneurysm clip and 5-0 braided silk suture. The heart was perfused retrogradely in a Langendorff system under constant flow (2.1 ml/min) by a Watson-Marlow (Falmouth, UK) peristaltic pump with 75 ml of a modified KH solution containing 10 mM 2,3-butanedione monoxime (BDM) and 10 μM blebbistatin (Cayman Chemical Company, Ann Arbor, Michigan, USA) (to give a KH-BDM/blebbistatin solution) to electromechanically uncouple the heart. The heart was then immediately transferred into ice-cold KH-BDM/blebbistatin solution. The atria were dissected from the rest of the heart, mounted onto Sylgard (Dow Chemical Company, Staines, UK) and placed in a bath containing filtered KH buffer solution. The latter was thermostatically maintained at 27 °C through all the experimental procedures performed.

aIIbb3 inhibition: Abciximab (C7E3-Fab) from Janssen and Tirofiban from Ibigen, myosin IIa inhibition: blebbistatin(−) (Cayman Chemical) (50 µM); blebbistatin(+) (Cayman Chemical) was used as a negative control. To avoid fluorescence-induced toxicity, para-nitroblebbistatin (50 µM, Optopharma) was used in a subset of experiments. Arp2/3 complex inhibition: CK666 (200 µM) and negative control: CK689 (both Merck Millipore). Actin polymerization inhibition: cytochalasin D (2.5 µM) (Sigma). Other: carboxyfluorescein succinimidyl ester (CFSE), Annexin V, busulfan, plasmin, thrombin, Fluo-4 AM, LPS O111:B4, CCL-2 (Sigma) iron (III) chloride (FeCl₃), casein, recombinant human albumin (HSA), fibrinogen from human plasma, unconjugated and conjugated to AlexaFluor 488 and 594, Rhodamine 6G, adenosine diphosphate (ADP), NP40, polyethylene glycol 400 (PEG400), phalloidin-AlexaFluor 488, hexamethyldisilazane (HMDS), paraformaldehyde (PFA), GDA, and LIVE/DEAD BacLight Bacterial Viability Kit were purchased from Sigma-Aldrich. Horm collagen was from Takeda, prostaglandin I2 sodium salt from Abcam, and U46619 from Tocris.

The PF573228, blebbistatin, or jasplakinolide (Cayman, Ann Arbor, MI, USA) were dissolved in DMSO. Then, 20 μL was applied once per day directly to the wound surface, starting at PWD 10 and continuing until PWD 17.

MEF cells were treated with 1% DMSO, 20 µM BAPTA (Cayman Chemical), Y27632 5 µM (Cayman Chemical), 50 µM CK666 (Cayman Chemical), STO-609 acetate (Biotechne, #1551), A23187 (Cayman Chemical), 4 µM Blebbistatin (Cayman Chemical), 40 uM AIP (R&D Systems #5959/1), 50 µM CAS 1090893 (Millipore, #553511), 0.1 µg/ml RhoA inhibitor-I (Cytoskeleton, Inc, #CT-04), 2 µM FK-506 (Cayman Chemical, # 10007965), 300 nM FAK14 (Cayman Chemical, #14485), 10 µM BMS-5 (Cayman Chemical, #21072), or 2 µM ML-7 (Cayman Chemical, #11801) overnight during time-lapse imaging for random cell migration or followed by immunostaining.

(±)-Blebbistatin (13186; Cayman, Ann Arbor, MI) was dissolved at 100 mM in DMSO and then diluted 2,000 times with culture medium. This Blebbistatin medium was then added to the chamber, to the bottom of which the cells had adhered, just after removal of the culture medium. After 30 min, the cells in the chamber were used for experiments without removal of the Blebbistatin medium.

After Control recordings, hearts were treated with 15 μM blebbistatin (Cayman Chemicals 13186) for 20 min and optical recordings were acquired as above. Next, hearts were treated with 4-AP (7 mM, Millipore Sigma 275875) and verapamil (1 μM, Sigma Aldrich V4629) one at a time, in the presence of blebbistatin. Optical recordings were once again acquired for each of these conditions as described above.