Versafluor fluorimeter

The mitochondrial membrane potential (ΔΨm) was determined by using the monitoring fluorescence of the specific probe 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3); (Sigma-Aldrich, Saint-Quentin Fallavier, France) as described previously [50 (link)]. DiOC6(3) differentially stained mitochondria as a function of their ΔΨm [51 (link)]. The cells were then incubated in the culture medium with 25 nM of DiOC6(3) at 30 °C for 45 min. After that, their fluorescence signal (λex = 482 nm and λem = 504 nm) was determined using the VersaFluor Fluorimeter (Biorad, Marnes-la-Coquette, France). The results were then expressed in the form of relative fluorescent units.
The metabolic activity of yeast cells was evaluated using the FUN-1 probe according to the manufacturer’s instructions (Molecular Probes, Illkirch, France) with previously described modifications [52 (link)].

The intracellular ROS/RNS level was assessed by using dihydrorhodamine-123 (DHR-123) fluorescent dye (Sigma-Aldrich, Saint Quentin Fallavier, France) as reported by Nazir et al. [32 (link)]. HepG2 cells in a pre-seeded 96-well plate (>90% viability; 1 × 10⁴ cells/well; 200 µL per well) were washed twice with phosphate-buffered saline (PBS), then resuspended in PBS containing 0.4 μM DHR-123 and incubated for 10 min at 30 °C in dark. Lastly, the fluorescence signal (λex = 505 nm, λem = 535 nm) was recorded on VersaFluor Fluorimeter (Biorad, Marnes la Coquette, France).

Yeast cells were first treated under the same conditions as mentioned above. Yeast cells were irradiated with a UV dose of 106.5 J/m2 UV-C (254 nm) under a Vilber VL-6.C filtered lamp (Thermo Fisher Scientific, Villebon-sur-Yvette, France), and incubated at 28 °C with orbital shaking at 120 rpm in the dark in complete 2.0% (w/v) glucose YPD medium (Sigma Aldrich, Saint-Quentin Fallavier, France) as previously described [65 (link)]. The same conditions were used to grow non-irradiated cells. Hour 0 of the oxidative stress experiment was considered irradiation.
The dihydrorhodamine-123 (DHR-123) fluorescent dye (Sigma-Aldrich, Saint-Quentin Fallavier, France) was used to assess the quantity of reactive oxygen and nitrogen species. Approximately 10⁸ yeast cells were washed twice in PBS, resuspended in PBS containing 0.4 M DHR-123, and incubated for 10 min in the dark at 28 °C in the presence of extract, RES, or DMSO (control cells). The fluorescence signal (ex = 505 nm, em = 535 nm) was measured using the VersaFluor Fluorimeter after two washes with PBS (Biorad, Marnes-la-Coquette, France).

Dihydrorhodamine-123 (DHR-123) fluorescent dye (Sigma-Aldrich, Saint-Quentin Fallavier, France) was employed to evaluate the reactive oxygen and nitrogen species (ROS/RNS) production levels as described previously [47 (link)]. For this, ca. 10⁸ yeast cells were washed twice using the PBS, resuspended in the PBS with 0.4 μM of DHR-123, and then incubated for 10 min at 30 °C in the dark. After performing the washing step 2 times using the PBS, the fluorescence signal (λex = 505 nm and λem = 535 nm) was then detected by using the VersaFluor Fluorimeter (Biorad, Marnes-la-Coquette, France).

For the experiment of protein extraction, approximately 10⁸ yeast cells were washed 3 times using the PBS. After that, 1 mL of the PBS was added, then the mixture was subjected to 3 freeze–thaw cycles using liquid nitrogen. The cell lysate was centrifuged at 10,000× g for 15 min at 4 °C, and the supernatant was then used to prepare the sample solution. The proteins were then quantified using a Qubit Protein Assay Kit according to instructions from the manufacturer, and using a Qubit fluorimeter (Thermo Scientific, Illkirch, France).
The total SOD activity was examined using a Superoxide Dismutase Activity Kit according to instructions from the manufacturer (Thermo Scientific, Illkirch, France).
The SIRT1/SIR2 activity was then investigated by using a SIRT1 Assay Kit (Sigma-Aldrich, Saint-Quentin Fallavier, France) according to instructions from the manufacturer, and using a Versafluor fluorimeter (Biorad, Marnes-la-Coquette, France).
Total NADH oxidase activity was assayed spectrophometrically at 25 °C in 50 mM of potassium phosphate buffer (pH 7.0), 0.29 mM NADH, and 0.3 mM EDTA [53 (link)]. A unit of activity was the quantity that catalyzed the oxidation of 1 μmol of NADH per min.

The NAD and NADH measurements were performed as described previously [48 (link)] using ca. 10⁷ cells. Briefly, after the extraction of NAD and NADH using the acidic and alkali extraction protocol, the concentrations were determined fluorometrically with the excitation at 365 nm and the emission at 460 nm by using the VersaFluor Fluorimeter (Biorad, Marnes-la-Coquette, France) with the standard curves (0–40 µM).
ATP was quantified as described previously [49 (link)] using the colorimetric ATP Assay Kit (Merck Millipore, Saint-Quentin Fallavier, France) according to the manufacturer’s instructions.