Polydatin

A total of 150 mice (20‐25 g) were randomly divided into the sham‐operated, SCI, PD‐treated, BMSC‐treated and PD+BMSC groups (N = 30 each; Figure S1B). Each animal was anaesthetized intraperitoneally with 2% (w/v) pentobarbital sodium (40 mg/kg), and the spinal cord was exposed by laminectomy at the T9‐T10 vertebral level. A contusion simulating thoracic SCI was produced using a pneumatic impact device in accordance with Allen's method.²³ The impact velocity was set at 0.5 m/s. The depth and duration of the impact were kept constant at 0.6 mm and at 80 ms, respectively. Postoperatively, the animals’ urinary bladders were manually voided twice daily. In the sham‐operated group, each mouse underwent a laminectomy, with no contusion injury performed. In the remaining groups, the mice were gastrically perfused with PD (20 mg/kg) once a day and/or transplanted with BMSCs at 5 days post‐injury (dpi) as appropriate. The surgical wound was opened, and a 3 μL suspension containing 2 × 10⁵ BrdU‐labelled BMSCs was injected into the injured site using a Hamilton syringe (Hamilton). The tip of the micropipette was kept in the spinal cord for 5 minutes after the injection. The mice in the SCI and PD groups were similarly injected with sterile PBS. After treatment, mice were killed and the spinal cords were extracted and stored at −80°C for subsequent experiments.

For LC-MS analysis of phenylpropanoid compounds, tomato fruit were harvested 7 days after breaker. The whole fruit pericarp was freeze dried and ground into a fine powder. An amount of 1 g of dry powder was extracted with 25 ml 80% MeOH at 4 °C under agitation, overnight. The sample was then centrifuged at 3,000 × g at 4 °C for 15 min and the supernatant was taken. The pellet was extracted again with 25 ml 80% MeOH at 4 °C for another 2 h. The supernatant was combined and further diluted 10 times with 80% MeOH. The samples were filtered through a 0.45 μm filter before injection. For each line, three biological replicates were analysed.
For flavonoid and isoflavone analysis, the samples were run on a Surveyor high-performance liquid chromatography (HPLC) system (Thermo) attached to a DecaXPplus ion trap MS (Thermo). Separation was on a 100 × 2.1 mm 2.6 μ Kinetex XB-C18 column (Phenomenex) using the following gradient of methanol versus 0.1% formic acid in water, run at 200  μl min⁻¹ and 30 °C: 0 min, 2% MeOH; 24 min, 38% MeOH; 30 min, 70% MeOH; 33.6 min, 70% MeOH, 34.2 min, 2% MeOH and 43.2 min, 2% MeOH. Data were collected for 34 min. UV-visible absorbance was collected with spectra from 200–600 nm from which chromatograms could be extracted at any wavelength, and a specific channel of 260 nm (bandwidth 9 nm), and positive electrospray MS spectra from m/z 150–2,000. The instrument also collected data-dependent MS2 spectra at an isolation width of m/z 4.0 and 35% collision energy, using dynamic exclusion to maximize the number of ions selected for fragmentation. Spray chamber conditions were 50 units sheath gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV. Sheath gas was supplied via the aux gas line and the entire flow from the LC passed through diode array to the mass spec ionization chamber without teeing.
Resveratrols were measured using a Surveyor HPLC system attached to a DecaXPplus MS (both Thermo). Separation was on a 100 × 2 mm 3 μm Luna C18(2) column (Phenomenex) running the following gradient of acetonitrile (ACN) versus 0.1% formic acid in water at 30 °C and 250 μl min⁻¹: 0 min, 2% ACN; 30 min, 70% ACN; 30.5 min, 2% ACN and 38 min, 2% ACN. resveratrol and polydatin were detected by UV absorbance at 306 nm (9 nm bandwidth) and by selected reaction monitoring in positive mode electrospray MS. The mass spec was set up to collect full spectra from m/z 150–1,000 and also targeted MS2 of precursor ion 229.0 at 40% collision energy and an isolation width of m/z 4.0, and precursor 391.1 at 40% collision energy and an isolation width of m/z 3.0. Spray chamber conditions were 50 units sheath gas, 5 units aux gas, 350 °C capillary temperature, and a spray voltage of 3.8 kV using a steel needle kit.

The warm water tail-flick test was used to determine pain threshold. Test was conducted before and during treatment. 4 cm of the rat-tail was placed in 50 ± 0.5°C warm water and the time between tail input and withdrawal from the water was recorded (three tests were conducted and the average in units of seconds was recorded). The latency was recorded with a sensitivity of 0.01 s. To perform a measurement, the rat had to remain calm, without unconditional unexpected movements of the tail. A maximum tail-flick latency of 10 s was used to minimize tissue damage to the tail (Liu et al., 2012 (link)).

We cultured fibroblasts derived from skin biopsies of three mitochondrial patients (P1, P2, and P3) harboring the following mutations:
We used control lines of primary human skin fibroblasts derived from healthy volunteer donors (C1, C2 and C3). These control cells were sex- and age-matched.
Patient and control cells were obtained following the Helsinki Declarations of 1964 (revised in 2001). Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco^TM, Waltham, MA, USA) with 10% Fetal Bovine Serum (FBS) (Gibco^TM, Waltham, MA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C and 5% CO₂. Cells were treated with polydatin and nicotinamide at 10 µM for seven days. All experiments were performed with cell cultures with a passage number lower than 10.

Drug screening was assessed by culturing the cells in a restrictive medium with galactose as the unique carbon source. The galactose medium was prepared using DMEM no glucose (Invitrogen^TM Molecular Probes, Eugene, OR, USA) supplemented with 20 mM D-galactose, 15 mM HEPES, 1% penicillin/streptomycin, and 10% FBS. Cells were seeded in 24-well plates in DMEM 1 g/L glucose and treated with different compounds. After 3 days, the glucose medium was removed and changed to galactose medium, with treatments reapplied. Images and cell counting were obtained immediately (T0) and 72 h after the shift to galactose medium (T72) using the BioTek^TM Cytation 1 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). The proliferation ratio was obtained by dividing the number of cells at T72 by the number of cells at T0. Proliferation ratio values above 1 were considered as cell proliferation, while values below 1 were considered as cell death, and a value of 1 indicated cell survival. Positive treatments were those that allowed the survival of patient cells in the glucose-free galactose medium, with the cocktail of polydatin and nicotinamide at 10 µM selected, as the others failed to make mutant cells survive and were deemed negative. Cell viability was confirmed by trypan blue dye exclusion.
This screening was repeated using 3-TYP, a specific inhibitor of SIRT3. The concentration employed was 32 nM, as this compound exhibits an IC₅₀ (half-maximal inhibitory concentration) of 16 nM for SIRT3, requiring a higher concentration for SIRT1 (IC₅₀ = 88 nM) and SIRT2 (IC₅₀ = 92 nM). This ensures the specific inhibition of SIRT3 without affecting other sirtuins. Cells were initially seeded in glucose medium and treated with polydatin and nicotinamide at 10 µM, along with 3-TYP at 32 nM for a duration of 3 days. Subsequently, the glucose medium was replaced with galactose medium, treatments were renewed, and images were captured using the same methodology as in the previous screening.