Caffeine

Ten animals were assigned to each of three groups based on their mean body weights to obtain an even distribution. Caffeine (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water (10 mL/kg) at concentrations calculated to deliver 120 and 180 mg/kg body weight/day (these Caffeine groups are designated CF1 and CF2, respectively) and administered by gavage to ensure complete consumption of the established daily dose in the morning (9 to 11 a.m.). The control group (CT) received distilled water daily for 4 weeks. The choice of dose levels was based on the literature, coupled with range finding studies to avoid sub-lethal effects at the highest dose [10 (link),16 (link)].
Animals were examined for any treatment-related clinical signs and weighed daily. Body weight was measured to the nearest 0.1 g with an electronic scale (Dretec Corp., Seoul, South Korea) and recorded from the day before the start of feeding of Caffeine for the four weeks of treatment. All the animals were killed 24 h after their last treatment, using established protocols and ethical procedures. Terminal blood samples were collected by heart puncture, and sera were stored at −70 °C.

Caffeine (Sigma) was dissolved in water at 80°C at a concentration of 100 mM. At 72 h after transfection with shRNA plasmids, cells were treated with 5 mM Caffeine diluted in medium for 2 h. At the end of the treatment, a 20-min pulse with 10 μM BrdU was performed. At this point, half of the cells were fixed in 75% ethanol for cell cycle analysis by BrdU/PI incorporation. Cells were incubated with UCN-01 (Sigma) at 500 nM for 2 h. A positive control for checkpoint activation was obtained by treatment of wt HeLa cells with 0.3 mM aphidicolin (Sigma) for 24 h.

Theobromine, caffeine, adenosine deaminase (ADA), and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) were from Sigma (St. Louis, MI, USA). SCH58261 was obtained from Tocris (Bristol, UK). The Aβ_1–42 peptide was purchased from Bachem (Bubendorf, Germany).
Theobromine was freshly prepared in milliQ water to a stock concentration of 1 mM. caffeine was prepared in milliQ H₂O to a stock concentration of 100 mM. Stock solutions of DPCPX (5 mM) and SCH58261 (5 mM) were prepared in dimethylsulfoxide (Sigma) and dilutions were prepared in ACSF or Krebs solution, controlling for the impact of the residual amount of dimethylsulfoxide. ADA, DPCPX, and SCH58261 were used in supramaximal but selective concentrations, respectively, 2 U/mL [66 ], 100 nM [67 (link)], and 50 nM [68 (link)]. Aβ_1–42 was dissolved in water to obtain a solution mostly composed of Aβ low molecular weight oligomers [19 (link),69 (link)].
All other chemical substances used, unless stated otherwise, were from Sigma (St. Louis, MI, USA).

For hypoxic experiments, the cell monolayers in culture dishes (6 × 10⁵ cells/dish) were transferred into a hypoxic chamber (Stemcell Technologies Inc., Vancouver, Canada) containing premixed gas (1% O₂, 5% CO₂, and 94% N₂) for 12 h. The temperature inside the chamber was set at 37°C, and the chamber was moistened with a dish containing sterile water. In parallel, the cells cultured in a humidified incubator with 21% O₂ and 5% CO₂ at 37°C served as the control (normoxia). To evaluate the effect of caffeine during hypoxia, the cells cultured under hypoxic condition were co-treated with various doses (3.125, 6.25, and 12.5 mM) of caffeine (Sigma–Aldrich).

Each food vial contained 1.5 g of Nutri-Fly^TM Instant Formulation (Genesee Scientific, San Diego, CA, USA) instant food (Genesee Scientific) and 7.5 mL distilled water, which swells to 10 mL volume. Caffeine (Sigma, St. Louis, MO, USA) was dissolved in distilled water and mixed with instant food to make supplemented food (Caffeine—0.25 mg/mL, 0.5 mg/mL, and 1.0 mg/mL).

The feeding performance of insects was evaluated using three feeding solutions. All solutions contained the phagostimulant ATP (0.001 M) to ensure the motivation of insects to feed. The following combinations were prepared: 1- low-NaCl (0.15 M); 2- high-NaCl (0.2 M); 3- caffeine (0.005 M caffeine in 0.15 M NaCl).
ATP was purchased from Sigma-Aldrich (St Louis, MO, USA), NaCl and caffeine from Biopack (Buenos Aires, Argentina).

Caffeine (#C0750, Sigma) was added to melted standard Drosophila media at a concentration of 0.5 mg/mL. At ZT0 on the day of sleep deprivation, flies were transferred into tubes containing the Caffeine-laced standard food. Following 24 hours of Caffeine supplementation, flies were then transferred back into tubes containing standard Drosophila media during the recovery period.