Dimethyl sulfoxide (dmso)

Cellular uptake assays using octadecyl rhodamine B chloride (R18)-labeled OMVs were performed as described previously (14 (link), 38 (link), 98 (link)). R18-labeled OMVs were diluted in cell culture medium depleted of FCS to a final concentration of 10 ng/μL based on quantification by Bradford assay. In the case of proteinase K treatment, MVs were incubated overnight at 55°C with 100 μg/mL proteinase K, followed by inactivation for 10 min at 65°C prior to the addition in cell culture. HT-29 or Caco-2 cells were seeded into black 96-well plates and incubated for 24 h at 37°C. Cells were washed with 200 μL medium depleted of FCS, and 1 μg of protein equivalent of MVs was added per well. In case of addition of uptake inhibitors, commercially available agents were added at the following concentrations: wortmannin, 0.1 μM in DMSO (Sigma-Aldrich); cytochalasin D, 0.5 μM in DMSO (Sigma-Aldrich); chlorpromazine, 0.35 μM in ddH₂O (Sigma-Aldrich); amiloride, 0.1 mM in DMSO (Sigma-Aldrich); nystatin, 0.2 μM in DMSO; (Sigma-Aldrich); and dynasore, 80 μM in ddH₂O (Sigma-Aldrich). Cells were incubated at 37°C, and fluorescence was measured for 8 h.

6 months old male zebrafish were exposed to either 0.01% dimethyl sulfoxide (DMSO, Sigma) and 0.1 or 1.0 μg/L E2 in 0.01% DMSO for 48 h, and 6 months old female fish were exposed to 0.01% DMSO and 0.1 or 1.0 μg/L 11-KT in 0.01% DMSO. Fish were randomly selected and divided into three groups (n = 10 per group). The treatment was performed in tanks [280 mm (L) × 210 mm (W) × 180 mm (H)] containing 2 L of the above solution. During the experiment, the solution was changed after 24 h. The 0.1 μg/L dose of E2 was selected because it has been shown to affect the reproductive system of male zebrafish^{45 (link), 46 (link)}. The corresponding 11-KT concentrations were selected to match and compare with E2 doses. During exposure, fish were fed an Adult Zebrafish Diet (Zeigler Bros.,) twice daily. After exposure, β-actin, bpifcl, kiss2 and gnrh3 expression was examined as described above. The primers used are shown in the Supplementary Table 1.

To examine the dose‐dependent effect of DMSO (NACalai Tesque, Kyoto, Japan), NPC seeded at 1.5 × 10⁴ cells/well in 96‐well plates or 5.0 × 10⁴ cells/well in 6‐well plates were exposed to various concentrations of DMSO ranging from 0.01 to 10% (v/v) prepared in αMEM supplemented with 20% (v/v) FBS for 1 and 24 h. To evaluate the time‐dependent effect of DMSO, NPC were exposed to 5% or 10% DMSO for 3, 6, 12, and 24 h. Furthermore, NPC were treated with a combination of 5% or 10% DMSO and 10 mM NAC (Sigma‐Aldrich) prepared in αMEM supplemented with 20% (v/v) FBS for 3, 6, 12, and 24 h. The concentration of NAC was based on previous work from Seol et al.⁵⁰ and confirmed most effective to retain cell viability (Figure S1). Cells exposed to 0% DMSO were used as an experimental control.

NEO212 is a crystalline powder. It was manufactured by Norac Pharma (Azusa, CA, USA) under current good manufacturing practice (cGMP) conditions, and by Axon MedChem (Groningen, The Netherlands). Both products were kindly provided by NeOnc Technologies (Los Angeles, CA, USA) and resulted in identical experimental results. NEO212 was dissolved in DMSO (Santa Cruz Biotechnology, Dallas, TX, USA) at 100 or 500 mM for in vitro or in vivo experiments, respectively. Temozolomide was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in DMSO to a concentration of 100 mM. Stock solutions of NEO212 and TMZ were stored at −80 °C. Perillyl alcohol was provided by NeOnc Technologies as NEO100 (Norac Pharma), which is a highly pure, cGMP-manufactured, pharmaceutical-grade version of POH that is in clinical use [33 (link)]. It is an oily liquid that was stored in a brown bottle at 4 °C. Immediately before use, it was diluted 1:12 with DMSO to produce a 600 mM stock solution. Further dilutions were carried out with cell culture medium as the diluent. O6-benzylguanine (O6BG; Santa Cruz Biotechnology) was dissolved in DMSO to 50 mM. 12-O-tetradecanoyl-phorbol-13-acetate (TPA; Sigma-Aldrich) was dissolved in DMSO to 20 mM. In all cases of cell treatment, the final DMSO concentration in the culture medium never exceeded 0.2%, and in fact was much lower in most cases.

Palytoxin was extracted from Palythoa Aff. Clavata following an established procedure (patent publication EP3087172B1). The compound, with a molecular mass of 2680.14 g mol⁻¹, was received as a powder, solubilized in DMSO (Sigma-Aldrich, Saint Louis, MO, USA), and further diluted to get working aliquots at 1 mM. Stocks and aliquots were stored at 4 °C for up to 2 years without loss of activity and protected from light and were used directly before the experiments. Etoposide was purchased from Sigma-Aldrich, Saint Louis, MO, USA, and dissolved in DMSO at a stock concentration of 50 mM. Pan-caspase inhibitor, z-VAD FMK, was purchased from Calbiochem (San Diego, CA, USA), dissolved in DMSO, and added 1 h before at a concentration of 50 μM. Protein phosphatase 2A inhibitor okadaic acid was purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO at a stock concentration of 1 mM. Proteasome inhibitor MG-132 was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in DMSO at a stock concentration of 10 mM. MAP kinase inhibitors, SB202190 and PD98059, were purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO at a stock concentration of 100 mM. Hydroquinone was purchased from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in DMSO at 10 mM. Inhibitors were used for 1h before palytoxin treatment at the indicated working concentrations.