2.2. In vitro assays

A total of 23 cancer cell lines, two noncancerous cell lines, one noncancerous primary cell and one mutant cell line and its parental control cell line were used in the study and are detailed in Table S1. The cells were cultured in flasks at 37°C in 5% CO₂ and were replicated every 3–4 days to maintain exponential cellular growth, following the instructions of the ATCC animal cell culture guidelines. A 0.25% trypsin EDTA solution (Sigma–Aldrich Co., Saint Louis, MO, USA) was used to remove adherent cells. All cell lines tested negative for mycoplasma, as evaluated by a mycoplasma stain kit (Sigma Aldrich Co.). All experiments were performed with cells at fewer than 50 passages.

The concentration of viable cells was determined by the trypan blue exclusion method. For that, 90 μL of the cell suspension was mixed with 10 μL of trypan blue (0.4%), and a haemocytometer was used to count viable (unstained cells) and nonviable (trypan blue‐stained cells) cells using a light microscope.

Cell viability was quantified by the Alamar blue assay. ¹⁵ Briefly, the cells were plated in 96‐well culture plates (30,000 cells/well for suspension cells or 7000 cells/well for adherent cells) and kept at 37°C in a 5% CO₂ atmosphere. BTZ was added to each well in duplicate and incubated for 72 h. Doxorubicin (purity ≥95%, Laboratory IMA S.A.I.C., Buenos Aires, Argentina) was used as a positive control. Four hours before the end of the incubation period (or 24 h for PBMCs), resazurin was added to each well at a final concentration of 3 μM. The absorbance values at 570 nm and 600 nm were evaluated using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Phenotyping of KG‐1a cells was performed using primary antibodies against CD11b, CD13, CD33, CD34, CD38 and CD123 conjugated with specific fluorochromes (Table S2). For that, the cells were rinsed with incubation buffer (0.5% bovine serum albumin in PBS) and incubated with antibodies for 1 h at room temperature. Then, the cells were rinsed with PBS, stained with YO‐PRO‐1 (Sigma–Aldrich Co.) and analysed by flow cytometry. A BD LSRFortessa cytometer using BD FACSDiva Software (BD Biosciences, San Jose, CA, USA) and FlowJo Software 10 (FlowJo LCC, Ashland, OR, USA) was used. At least 30,000 events were analysed per sample. Cell doublets and debris were excluded from the analyses.

The localization of NF‐κB p65 was investigated by confocal microscopy. Briefly, cells were washed twice with PBS, plated as droplets (5 μL) on coverslips, permeabilized with Triton X‐100 (0.5%), treated with RNase (10 μg/mL), washed with PBS and incubated overnight with an anti‐NF‐κB p65 antibody (Table S2 contains antibody details). The following day, the cells were washed with PBS and mounted with Fluoromount‐G (Invitrogen, Thermo Fisher Scientific) containing DAPI. The cells were photographed using a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, HE, Germany).

Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen; Hilden, Germany) according to the manufacturer's instructions. RNA purity was examined and quantified using a NanoDrop® 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA reverse transcription was evaluated using the Superscript VILO™ kit (Invitrogen Corporation; Waltham, MA, USA). qPCR analysis of gene expression was performed on a TaqMan® Array Plate 96 plus fast (#4413256, Applied Biosystems™, Foster City, CA, USA) on an ABI ViiA7 system (Applied Biosystems™). The PCR cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative quantification (RQ) of mRNA expression was calculated using the 2^−ΔΔCT method ¹⁶ on Gene Expression Suite™ software (Applied Biosystems™). Cells treated with 0.2% DMSO (negative control) were used as calibrators. The geometric mean RQs of the three reference genes GUSB, HPRT1 and GAPDH were considered for data normalization. All experiments were performed under DNase/RNase‐free conditions. A gene was upregulated if its RQ ≥2. Similarly, genes were downregulated when RQ ≤0.5.

Internucleosomal DNA fragmentation and cell cycle progression were assessed by flow cytometry, which detected DNA content with propidium iodide (PI). ¹⁷ The cells were stained with a solution containing 0.1% Triton X‐100, 2 μg/mL PI, 0.1% sodium citrate, and 100 μg/mL RNase (all from Sigma Aldrich Co.). The cells were analysed by flow cytometry after 15 min of incubation in the dark, as described above. At least 10,000 events were examined per sample.

Apoptotic cells were detected by annexin V‐FITC/PI (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences) or YO‐PRO‐1/PI (Sigma–Aldrich Co.) according to the manufacturer's instructions. The cells were analysed by flow cytometry, as described above. At least 10,000 events were analysed per sample. The antioxidant N‐acetylcysteine (NAC) and the pancaspase inhibitor Z‐VAD(OMe)‐FMK were used in the functional assays.

Mitochondrial transmembrane potentials were evaluated using cells stained with rhodamine 123. ¹⁸ After 24 h of treatment, the cells were stained with 1 μg/mL rhodamine 123 (Sigma–Aldrich Co.) and incubated for 15 min at 37°C in the dark. After that, the cells were rinsed and analysed by flow cytometry, as described above. At least 10,000 events were analysed per sample.

The levels of active caspase‐3 and cleaved PARP (Asp 214) were quantified using primary antibodies conjugated with specific fluorochromes (Table S2) following a protocol for intracellular staining of cells. The cells were collected and resuspended in 0.5–1 mL of 4% formaldehyde for 10 min at 37°C. The tube was then placed on ice for 1 min. Cells were permeabilized on ice for 30 min by slowly adding ice‐cold 100% methanol to prechilled cells with gentle vortexing until the final concentration of methanol reached 90%. After washing with an incubation buffer (0.5% bovine serum albumin in PBS), primary antibodies were added and incubated for 1 h at room temperature. Then, the cells were rinsed with PBS and analysed by flow cytometry, as described above. At least 10,000 events were analysed per sample.

Mitochondrial superoxide levels were quantified using MitoSOX™ Red reagent (Thermo Fisher Scientific, Waltham, MA, USA), and analysis was performed according to the manufacturer's instructions. The cells were analysed by flow cytometry, as described above. At least 10,000 events were analysed per sample.