N3876

CVE was performed using a vacuum-based vapor exposure system from La Jolla Alcohol Research^{17 (link),19 (link),20 (link)}. Animals were placed into sealed home cages with their standard housing cage-mates for 8 h a day, 5 days a week (9:00–17:00 M-F) before being returned to their standard home cages. The sealed cages continuously received clean room air from a down-stream vacuum pump (16 L/m), and a computer-controlled Electronic Nicotine Delivery System (ENDS) delivered vapor (3 s, 200 °C) at pre-defined inter-vape intervals (2, 5, 10, 15, and 60 min). The e-liquid vehicle (VEH) consisted of 50% propylene glycol (Sigma-Aldrich, P4347) and 50% vegetable glycerin (Sigma-Aldrich, G5516) (50/50 PGVG), and all experiments with nicotine (NIC) used a dose of 20 mg/ml (−)−nicotine (Sigma Aldrich, N3876). During all vapor exposure procedures, mice could move freely within the home cage with ad libitum access to food and water.

We used the same assay described in Marriage et al. (2014) (link), except here we tested larvae in narrow polystyrene vials rather than in plates. Briefly, flies were allowed to lay eggs on a cornmeal–yeast–molasses medium containing 0.5% activated charcoal, supplemented with a small amount of active yeast paste to elicit egg laying. First instar larvae were collected and placed either on no-drug media or on media containing 0.18 μl/ml nicotine (N3876; Sigma). No-drug and nicotine media were always prepared the day prior to larval collection to minimize variation due to nicotine breakdown. Replicate assay vials contained 30 first instar larvae in nearly all cases (Supplemental Material, File S1), and the phenotype for each replicate vial is given as the fraction of larvae that ultimately emerge as adults. For every genotype tested, we set up several egg-laying vials, and the mean phenotype is given as the average of multiple replicate assay vials.
All animals were reared and tested at 25° and 50% relative humidity on a 12-hr-light/12-hr-dark cycle. Those test animals that were the result of crosses were generated by pairing 10 virgin females with four to five males over several replicate vials. Prior to initiating such crosses, parental flies were allowed to recover from CO₂ anesthesia for 24 hr.

E-liquid RM was prepared by combining propylene glycol, glycerin and nicotine. Propylene glycol (Sigma Aldrich 49770) and glycerin (Sigma Aldrich 82280) were puriss. p.a., analytical grade chemicals that met American Chemical Society (ACS) specifications. They were tested to be >99.9% pure by Gas Chromatography (GC) and can be traced by Lot Numbers SZBE279CV and BCBN5225V, respectively. Nicotine solution (Sigma Aldrich N3876) was tested to be 99.5% pure (Lot Number 1449194 V) (Fig. 1).Fig. 1

A Standard Operating Procedure (SOP) for e-liquid Reference Material formulation by adopting ISO Guide 35:2006E (Reference Materials: General and Statistical Principles for Certification) guideline. The reference e-liquid was characterized by measuring quality, homogeneity, stability and traceability of each component of the e-liquid

(−)-Nicotine (N3876; Sigma-Aldrich, St. Louis, MO) and (−)-cotinine (C5923; Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO). ARPE-19 cells or RPE explants were treated with 1 or 2 μM nicotine, cotinine or nicotine-cotinine mixture in DF-12 culture medium (final concentration of DMSO was 0.1%) for 5–7 days. The control group was the cells treated with 0.1% DMSO in DF-12 culture medium. The medium was changed daily in order to maintain a constant level of nicotine and cotinine.

The following drugs were used: atropine (Sigma-Aldrich #A0132), nicotine (Sigma-Aldrich #N3876), muscarine (Sigma-Aldrich #M6532), acetylcholine (Sigma-Aldrich #A6625) and TTX (Alomone Labs #T-550). In all cases, stock solutions were prepared were diluted to the final concentration before experiments. Drugs were applied either by bath application or were injected directly to the AL using a pico-injector (Harvard Apparatus, PLI-100).

Four-week-old male BALB/c mice, purchased from the Shanghai Laboratory Animal Center (SLAC), China, were inbred under a specific pathogen-free environment at the Animal Experiment Center of Wenzhou Medical University. All experiments were performed with the approval of the Wenzhou Medical University Ethics Committee and in accordance with the China Animal Welfare Legislation, as well as the guide for the care and use of laboratory animals. The mice were randomly divided into five groups: the normal control group (NC), the viral myocarditis group (VMC), the nicotine (Sigma-Aldrich, N3876, 0.2 mg/kg/d, i.p.) treatment group, the nicotine and α-Conotoxin AuIB [18 (link)] (Tocris Bioscience, 700 pmol/mouse/d, i.p.) treatment group, and the nicotine and LY294002 (Selleck Chemicals, 0.4 mg/kg/d, i.p.) treatment group. 1.0 × 10⁶ plaque-forming units of purified CVB3 (Nancy strain) in 0.1 ml phosphate-buffered saline (PBS) were i.p. injected in the mice from the VMC group and the treatment groups on the 1st day of the experiment. Normal controls received PBS instead of CVB3. Animals of each group (n ≥ 8) were sacrificed at day 7 postinfection, and hearts were snap-frozen and stored at −80°C for analysis.

Female mice (n = 9) were administered nicotine (Sigma, N-3876; 200 μg/ml) in drinking water containing 1% saccharin (Shyuanye; 128-44-9) starting from 2 weeks premating until the offspring were weaned. The female control mice (n = 9) received drinking water containing 1% saccharin. Two weeks after drinking nicotine water, all female mice were paired with male mice at a 3:1 ratio until they gave birth. All female mice gave birth to babies. The offspring were weaned on postnatal day 20 (P20) and distributed randomly into experimental groups (Supplementary Table 1).