Veratridine

After marking the areas to be analyzed, each tissue sample pinned on Sylgard was imaged by D-FFOCT before treatment (T0). Sample was then removed from the carrier plate, placed in 6-well cell culture plates and treated with 75 µM of veratridine (Sigma-Aldrich, Saint Louis, USA), 1 µM of TTX (Bio-Techne SAS, Noyal-Châtillon sur Seiche, France), 100 mM, 200 mM, and 300 mM of D-Mannitol (Sigma-Aldrich), 0.1% (vol/vol) DMSO (veratridine control) or Krebs (TTX and Mannitol control) for 30 min at room temperature under agitation (200 rpm). Then, each treated-tissue sample pinned on Sylgard was returned to the scanner holder with the same pressure and localization (X, Y) to be imaged by D-FFOCT (T1). Each tissue sample was then washed twice 15 min each and imaged again by D-FFOCT (T2).

In this study, we used pLKO.1 control and DJ-1-deficient SH-SY5Y neuron-like cells previously generated
by our group.^{29 (link)} Cells were cultured at
37 °C and 5% CO₂ in selective growth medium consisting
of Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)
(Biowest) and supplemented with 10% (v/v) fetal bovine serum (Capricorn),
1% non-essential amino acids, 100 mg/mL penicil/streptomycin (Labclinics),
and 2 μg/mL puromycin (Labclinics). Viability of cells treated
with VIN, the VNGC activator veratridine (Santa Cruz Biotechnology,
sc-201075), or 0.1% DMSO (vehicle) was evaluated using a MTT assay
(Sigma-Aldrich) as described in.^{29 (link)} To evaluate
if veratridine could impair the beneficial effect of our candidate
compound, cells were pretreated for 2 h with 150 μM of the VNGC
activator before the addition of VIN. Subsequently, viability assays
were performed as described in ref (29 (link)). All experiments were carried out using three
biological replicates and three technical replicates per sample.

mBMMCs were loaded with 3 µM Fluo-8 AM (AAT Bioquest) for 30 min. After washing with Kreb’s solution, mBMMCs were added to a dish of isolated myenteric neurons for an enteric neuron–immune cell coculture system. Then, [Ca²⁺]i in mBMMCs was measured as fluorescence intensity with MiCAM02. Neurostimulator veratridine (Na⁺ channel activator; 10 µM; Sigma-Aldrich), for stimulation of only isolated myenteric neurons, was added to a dish at 20 s, and calcium ionophore A23187 (10 µM) was added to the dish at 250 s after starting the measurement for maximum stimulation of mBMMCs. The activation rate was calculated by the following formula: (the highest fluorescent intensity from 20 s to 249 s/the highest fluorescent intensity from 250 s to 500 s) × 100. Selective adenosine A3 receptor antagonist MRE 3008F20 (R&D systems) at 10 μM was applied to the dish 20 min before the administration of veratridine.

Cells were purchased from American Type Culture Collection (VA, USA) and they were grown in the recommended medium. Veratridine, Veratrine, etoposide, 5-Fluorouracil and staurosporine were purchased from Sigma (MO, USA). Because we used DMSO as a solvent for VTD at different concentration (10–300 μM), we examined different concentrations of DMSO (0.02%–0.3%) corresponding to VTD used (10–300 μM) in HCT-116 cells. As the results show in Panel D of Supplementary Figure S1, concentrations of DMSO as a vehicle had no cytotoxic effect on HCT-116 colon cancer measured by MTT assay at 24, 48, and 72 hours. This set of data further confirmed the cytotoxic effect observed with VTD is purely related to the anti-cancer function of VTD and not DMSO.