Colchicine

For chronic colchicine treatment of mice, 8–12-week-old male C57BL/6 wild-type mice were randomly assigned to vehicle, colchicine, PE, and colchicine/PE treatment groups. colchicine treatment followed a modified protocol from refs. ^{10 (link)} and ^{36 (link)}. Briefly, mice were I.P. injected every other day with colchicine in increasing doses (0.5, 0.75, and 1 mg/kg body weight) to improve tolerance^{9 (link)} and were maintained at the 1 mg/kg BW dose. Control animals were I.P. injected with PBS. PE (10 mg/kg BW in 0.2% ascorbic acid (Sigma-Aldrich) dissolved in PBS) was administered subcutaneously ~0.5–1 h after colchicine treatment (to ensure sufficient colchicine accumulation in the myocardium at the time of PE action²⁰ ) for 3 total injections over 5 days. Control animals received subcutaneous injections of vehicle (0.2% ascorbic acid in PBS). Mice showed no overt signs of distress or toxicity during this protocol, and on day 9 were euthanized for tissue harvesting. For acute colchicine treatment of rats, rats were injected I.P. with 1 mg/kg body weight sterile colchicine (Sigma-Aldrich) dissolved in PBS. The animals were euthanized 16–20 h after injection with colchicine.

Sprague-Dawley WT rats (n = 24, male, 300 ± 20 g) were purchased from “Cantacuzino” National Medico-Military Institute for Research and Development, Bucharest, Romania.
NIH (National Institute of Health) guidelines were followed for all procedures. Animal Ethics Committee of the University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj-Napoca approved the experiment (Permit number: M20150505).
Animals were kept in standard conditions (room temperature 23 ± 1 °C and 55 ± 5% relative humidity, 12 h light/dark cycle). Food and water were provided ad libitum.
Colchicine used in the experiment was purchased from Sigma-Aldrich, USA, batch number: SLBQ8131V.
The types of diet used were basic diet (BD) and high-fat diet (HFD) purchased and prepared as previously described [25 (link)].
Rats were included randomly into three groups: (1) WT + BD group (wild type rats fed with a standard diet, n = 8); (2) WT + HFD (wild type rats fed with HFD, n = 8); (3) WT + HFD + Col (wild type rats fed with HFD and treated with Colchicine 0.5 mg/kg, intraperitoneally (i.p), daily administration, n = 8) [26 (link)].
The total duration of the study was nine weeks and during this time rats received either standard or HFD, while after four weeks of HFD, Colchicine treatment was started, for 5 weeks.
After 9 weeks, blood, liver, aorta and myocardial tissue were sampled, as previously described [25 (link)].

Assessment of the impact of colchicine and paclitaxel on cells of A. islandica was performed via progressive addition of colchicine (Sigma-Aldrich) or paclitaxel (Sigma-Aldrich) to the medium to a final concentration of 5, 10 or 20 µg ml⁻¹ for colchicine, and 0.1, 1, 10 or 100 µM for paclitaxel. Lysotracker Yellow H-123 viable dye (Thermo Fisher Scientific) was added simultaneously to a final concentration of 0.3 µМ (Declés et al., 2008 (link)). All experiments were conducted in triplicate.