**2. Material and Methods**

*2.1. CUR and DSS Obtaining*

CUR and DSS were purchased from Sigma-Aldrich (Sigma-Aldrich Co., Saint Louis, MO, USA).

#### *2.2. In Vitro Assays*

#### 2.2.1. Cells

The mouse melanoma B16-F10 cell line and human lung fibroblast MRC-5 cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured as recommended by the ATCC animal cell culture guide. All cell lines were tested for mycoplasma using a mycoplasma staining kit (Sigma-Aldrich Co.) and were free from contamination. Cell viability was examined using the trypan blue exclusion method for all experiments. Over 90% of the cells were viable at the beginning of culture.

#### 2.2.2. Alamar Blue Method

The quantification of cell viability was carried out by the Alamar Blue method, as previously described in refs. [30–32]. Briefly, the cells were seeded in 96-well plates and incubated for 72 h. CUR and DSS were tested on a concentration-response curve obtained by serial dilution of a stock dissolved in dimethyl sulfoxide (DMSO; Vetec Química Fina Ltd.a, Duque de Caxias, RJ, Brazil), with concentrations based on previous studies [33–35]. Doxorubicin (DOX, doxorubicin hydrochloride, purity ≥ 95%, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) was used as a positive control. At the end of the treatment, 20 μL of a stock solution (0.312 mg/mL) of resazurin (Sigma-Aldrich Co.) were added to each well. Absorbances at 570 and 600 nm were measured using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

#### 2.2.3. Flow Cytometry Assays

To quantify cell death, the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) was used, and the analysis was performed according to the manufacturer's instructions. Cell fluorescence was determined by flow cytometry.

DNA fragmentation and cell cycle distribution were determined using 2 μg/mL propidium iodide (PI) in cells permeabilized with 0.1% Triton X-100, 0.1% sodium citrate and 100 μg/mL RNAse (all from Sigma-Aldrich Co.), as previously described in ref. [36], and cell fluorescence was assessed by flow cytometry.

Detection of superoxide levels was performed using MitoSOX™ Red reagent, a fluorogenic dye specifically targeted to mitochondria in living cells (Thermo Fisher Scientific, Waltham, MA, USA), and the analysis was performed according to the manufacturer's instructions. Cell fluorescence was determined by flow cytometry.

For all analyses using flow cytometry, 10,000 events were recorded per sample with a BD LSRFortessa cytometer and analyzed with BD FACSDiva Software (BD Biosciences, San Jose, CA, USA) and FlowJo Software 10 (Flowjo LCC, Ashland, OR, USA), and cellular debris was omitted from the analysis.
