Male nmri mice

Male NMRI mice (weight 18–20 g) were acquired from Charles River Laboratories (France) and housed in polyacrylic cages under a 14/10-h light/dark cycle at 21 °C. The animals were fed a pellet diet and water ad libitum and were allowed to acclimatize for one week before experimental procedures were conducted. Prior to the experiment, mice were isolated in polyacrylic cages with a pellet diet and water ad libitum for habituation overnight in the experimental room, to minimize stress.

Male NMRI mice (Charles Rivers) were inoculated at 8 weeks of age with 1 × 10⁷ CWR22RV1 cells with GCNT1 knockdown by unilateral subcutaneous injection into the flank. Cells were injected in a volume of 100 µL and Matrigel in a 1:1 mixture. Animals were weighed and tumour volumes monitored by caliper measurement three times a week until the first animal met a humane endpoint.

All animal care and use were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NRC) and experimental protocols were approved by the Institutional Ethical Committee on Animal Experimentation, according to applicable regional law. Male NMRI mice were purchased from Charles River Laboratories (France), male wild-type FVB mice were purchased from Janvier (France), and male transgenic Gfap-luc mice (FVB/N-Tg(Gfap-luc)-Xen) were obtained from Taconic Laboratories (USA). The latter animals express luciferase under the transcriptional control of the glial fibrillary acidic protein (Gfap) promoter [32 (link)] and are commonly used as a model system for noninvasive quantification of astrocyte activation in living animals over time [27 (link), 33 (link), 34 (link)]. Unless mentioned otherwise, animals were housed in groups of 4 per cage under a normal 12:12 h light-dark cycle (lights on at 06:00 a.m. with a 30 min dim and rise phase). Food and water were available ad libitum.
Recombinant mouse TNF-α was purchased from Biolegend (product ID 575208) and dissolved in sterile phosphate buffered saline prior to injection.

Male NMRI mice (8–12 weeks old and 25–35 g body weight; Charles River, Sulzfeld, Germany) were used. NMRI mice were selected as this strain has displayed robust behavioural responses to nicotine in our previous studies, as reflected by activation of the mesolimbic dopamine system (Jerlhag and Engel, 2011 (link); Egecioglu et al., 2013 (link)). It should however be mentioned that other strains of mice, as well as rats, show similar behavioural responses to nicotine (Tzschentke, 1998 (link); Jerlhag and Engel, 2011 (link); Natarajan et al., 2011 (link); Egecioglu et al., 2013 (link); Ignatowska-Jankowska et al., 2013 (link)) and similar effects to sCT (Kalafateli et al., 2020b (link)). The mice were group-housed and maintained at a 12/12-h light/dark cycle; they acclimatized to the animal facility (temperature of 20°C with 50% humidity) one week before experiments. They had ad libitum access to water and standard chow (Teklad Rodent Diet; Envigo, Madison, WI, United States) before and after each experiment. An independent set of age-matched mice was used in each behavioural experiment. For the Western Blot experiments, the mice brains from the repeated sCT-nicotine locomotor sensitisation test were used. All experiments were approved by the Swedish Ethical Committee on Animal Research in Gothenburg (207–2014; 195–2014; 1,457–2018; 3,348–2020).

Male NMRI mice (12 weeks old) were obtained from Charles River and housed under specific pathogen-free conditions with the approval of the local animal ethics and welfare committee. The animals were maintained in a 12-h light–dark cycle and were provided with food and water at libitum. Animal experiments are in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (OJ of EC L 358/1 12/18/1986) and ARRIVE guidelines. This study was approved by the University of Messina Review Board for the care of animals in compliance with Italian regulations on protection of animals (n 499/2018-PR released on 23 February 2018).