C1386

When the tumors were about 1 cm, rats were treated with curcumin (C1386; Sigma Aldrich, MO, USA) and electrical pulses (EP). Figure 1b shows the equipment and procedure used for the treatment. Curcumin was first dissolved into DMSO (BP231-100; Fisher scientific, MA, USA) and was diluted further in saline to make 200 µM concentration for injections. First, curcumin injections (100 µL) were administered intratumorally. After 1 min of curcumin injection, multiple EP (1000 V/cm, 100 µs, 8 pulses at 100 ms interval between pulses) were administered using needle array electrodes (Leroy Biotech, France) composed of two rows of 4 needles (15 mm long, 2.9 mm gap between needle tips and 5.9 mm gap between rows) to cover the entire tumor area. The BTX ECM 830 electroporator (BTX, MA, USA) was used for generating EP. The rats were anesthetised during the procedure. When the mammary tumors reached 2.5 cm in any one dimension, rats were euthanized.

Treatment of primary isolated SCs with curcumin was performed by mixing the compound in the culture medium. The starting point was a 10 mM curcumin stock solution (Cat#: C1386, Sigma-Aldrich Taufkirchen, Germany) diluted in 100% EtOH, as reported in the literature [61 (link),75 (link),76 (link),77 (link)]. This stock solution was stored at −20 °C until use. All subsequent dilutions were performed with a culture medium.

Dye-binding assays were performed essentially as described previously [44 (link)]. Samples containing 5 µM Aβ aggregates were prepared as indicated above. Dyes were added to the samples at a concentration of 5 µM for curcumin (Sigma-Aldrich #C1386) or 4 µM for hFTAA, and then samples were incubated for 15 min at room temperature with shaking (850 rpm). To remove unbound dye, samples were placed in Slide-A-Lyzer Mini dialysis devices with a molecular weight cutoff of 10 kDa (Thermo Scientific #69570) and dialysed against dH₂O for ~ 50 min. After dialysis samples were recovered and placed in a half-area black clear-bottom 96-well microplate (Greiner Bio-One #675096). Fluorescence emission spectra were measured using a BMG CLARIOstar microplate reader. For curcumin, an excitation bandwidth of 432 ± 7.5 nm was used and fluorescence emission values from 460 ± 5 to 625 ± 5 nm were measured. For hFTAA, an excitation bandwidth of 488 ± 5 nm was used and fluorescence values from 513 ± 5 to 690 ± 5 nm were measured. Background signal from reactions containing only dye were subtracted, and then fluorescence signals were normalized to the highest value obtained, which was set at 1.0.

Caffeine (1,3,7-trimethylxanthine) (HiMedia: RM6753), curcumin (diferuloylmethane) (Sigma: C-1386), and naringenin (4′,5,7-trihydroxyflavanone) (Sigma: N-5893) were all diluted to 10 mM stock solutions with deionized water just prior to use. To avoid the radical scavenging activity of solvents such as DMSO or ethanol, addition of a small amount of NaOH (20–100 μL of 1 M NaOH per 10 mL polyphenol stock solution) was sufficient to quickly and completely dissolve curcumin and naringenin. Stock solution of hydrogen peroxide [10 mM] was prepared fresh in PBS solution [10 mM] and copper sulphate [10 mM] was dissolved in water before use. The phytochemicals were stored in 4°C, and wrapped with aluminum foil to reduce their sensitivity to light.

Stock solutions of doxazosin (D9815; Sigma) and carvedilol (PHR1265; Sigma) were prepared at a concentration of 50 mM using DMSO as a solvent. Based on this solution, the preset treatments at concentrations of 0.1, 0.3, 0.5, 10, 15, and 25 μM were obtained using complete DMEM as a solvent. A 1 mg/mL stock solution of curcumin (C1386; Sigma) in 1% DMSO was prepared. Subsequently, a dose-response curve was elaborated using successive dilutions of the stock solution (0.01, 0.03, 0.05, 0.07, 0.1, 0.5, 1, 5, 10, and 25 μM/mL) and evaluating the viability of the HepG2 cells using the MTT assay, to apply 1 h before the interaction with doxazosin and carvedilol.

Nanoparticles were prepared by Dr. Antonio Claudio Tedesco (Department of Chemistry, University of Sao Paulo at Ribeirão Preto). Curcumin was obtained commercially (Sigma-Aldrich Co. cat# C1386, Lot# 081M1611V). A 1:1 ratio of poly-lactic and polyglycolic acids (obtained from Sigma-Aldrich Co.) were loaded with 0.05 mg/mL of curcumin (ethanol solution). The vehicle control (‘empty’ nanoparticles) were prepared using the same method, but using loaded only with the same volume of ethanol vehicle. Briefly, the polymers were dissolved in di-chloromethane and added to a 1% acqueous solution containing polyvinyl alcohol and curcumin. The mixture was submitted to vigorous agitation for emulsification of water in oil. The organic solvent was removed by agitation at room temperature followed by evaporation under reduced pressure. The particles were centrifuged (1,100–4,600 g, 4 C) and, after three washes with distilled water, resuspended in PBS and filter-sterilized. Nanocurcumin and the empty nanoparticle vehicle were stored protected from light at 4 C and used within 30 days of its preparation.

EpASCs from neonates born to mothers in control group were employed for the assay. Cells were seeded (2*10⁴ cells/well) in 96-well plates and allowed to adhere for 24 hours in a CO₂ incubator. iAs and selenite were dissolved in MilliQ water; curcumin was dissolved in DMSO. Final concentration of DMSO in the assay was ≤0.05%; flasks from the control group received only the vehicle. Cells were exposed (in triplicate, for 24 hours) to serial dilutions (40 to 1.25 μM) of arsenite (NaAsO₂, Sigma catalogue #S7400) or selenite (Na₂SeO₃, Sigma catalogue #S5261) or curcumin (Sigma catalogue #C1386). After 24h exposure, MTT (10μl of 5mg/ml) was added and cells were further incubated for 3-4hrs. The formazan crystals formed in viable cells were dissolved in DMSO. A₅₄₀ was determined using a microplate reader (Microquant, Bio-Tek, USA) to establish EC₅₀ value of arsenite, selenite and curcumin (S1a Fig).