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  • Phytomedicine br NaCl was purchased from Jos Manuel


     Phytomedicine 63 (2019) 153017
    NaCl was purchased from José Manuel Gomes dos Santos, Lda. Lactacystin, 20S proteasome and Suc-Leu-Leu-Val-Tyr-AMC (20S protea-some fluorogenic substrate) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Hydrochloric Hexa His tag peptide was from VWR International, LLC. Ac-Leu-Glu-His-Asp-7-Amino-4-trifluoromethylcoumarin (caspase-9 substrate) was purchased from CPC Scientific (Sunnyvale, CA, USA). Anti-CHOP Antibody was purchased from Thermo Scientific. β-Tubulin primary antibody, as well as anti-rabbit secondary antibody, were from Santa Cruz Biotechnology (Dallas, TX, USA). Z-Leu-Glu-Val-Asp-AFC (caspase-4 sub-strate) was obtained from Innovagen (Lund, Sweden). FITC Annexin V and 7-AAD Viability Staining Solution were purchased from BioLegend. Kapa Sybr® Fast was acquired from KapaBiosystems. PureZol™ RNA Isolation Reagent was acquired from BioRad.
    Cell culture
    Assessment of viability - MTT assay
    A549 cells were plated at a density Hexa His tag peptide of 1 × 104 cells/well, AGS at 1.5 × 104 cells/well and MRC-5 at 2 × 104 cells/well, followed by in-cubation for 24 h under the conditions described above, like reported before (Pereira et al., 2015b). After an incubation period of 8 or 24 h with the molecules, 0.5 mg/ml MTT solution was added and incubated for 2 h. The anticancer molecule irinotecan hydrochloride was used as positive control. The formazan in each well was dissolved in a solution of 3:1 DMSO:isopropanol. Lastly, the absorbance at 560 nm was read in a Thermo Scientific ™ Multiskan ™ GO microplate reader.
    Assessment of membrane integrity - LDH release assay
    The extracellular amount of LDH was evaluated as a strategy to assess membrane integrity as described before (Pereira et al., 2014b; Silva et al., 2017). The cells were incubated in the presence of each compound. After this, LDH was measured into the culture media su-pernatant by monitoring the decrease of NADH during the conversion of pyruvate to lactate, at 340 nm in microplate reader (Multiskan AS-CENT, Haverhill, MA, USA). For positive control for cell lysis was used triton X-100 1%.
    Flow cytometry
    For flow cytometry, cells were cultured in 6-well plates
    (3.6 × 105 cells/ml) and treated with the different benzoquinones (cyperaquinone and hydroxycyperaquinone at 6.25 μM), during 8 h at 37 °C. Cells treated with staurosporine (0.5 μM) (Sigma–Aldrich Co., Saint Louis, USA) for 18 h were used as positive control. After incuba-tion, cells were trypsinized, washed with PBS and incubated with Annexin-V/7-AAD (0.09 µg/µl and 0.05 μg/µL respectively) for 10 min. Bivariant analysis of Annexin-PE fluorescence (FL-1) and 7-AAD fluorescence (FL-3), based on the acquisition of 40 000 events/cells in BD Accuri™ C6 cytometer (San Jose, CA, U.S.A), distinguished different cell populations, Annexin V−/7-AAD− were considered as viable cells, Annexin V+/7-AAD− corresponded to apoptotic cells and Annexin V +/7-AAD+ were designated necrotic cells. Detectors for the three fluorescent channels (FL-1, FL-2 and FL-3) were set on a logarithmic scale, while FSC and SSC light scatter were set on a linear scale. For the analysis using BD Accuri™ C6 analysis software, the 7-AAD+ population were gated out (necrotic cells), being only analysed Annexin V-/7-AAD− (viable cells) and Annexin V+/7-AAD− (apoptotic cells) populations.
    Morphological analysis
    Cells were cultured in coverslips at a density of 5 × 104 cells/well in 24-well plates in the presence of the molecules under study. Cells were incubated for 24 h and, after that, they were washed with HBSS and fixed with cold methanol for 30 min at 4 °C. Phalloidin–tetramethylrhodamine B isothiocyanate (0.5 µg/ml) and DAPI (0.1 µg/ml) were added and cells were stained for 1 h at room temperature.
    Intracellular ROS levels
    Caspases activity assay
    The activity of caspase-3 was evaluated using the Caspase-Glo® 3/7 kit assay. Cells were incubated with the compounds and staurosporine (positive control) according to the above-mentioned conditions for the MTT assay in 96-well white plates, at 37 °C, for 8 h. Caspase-Glo® 3/7 buffer and Caspase-Glo® 3/7 substrate were added to the same volume of cell supernatant and incubated for 35 min at 22 °C. After that, the luminescent signal was measured in a microplate reader (Cytation™ 3, BioTek, Winooski, VT, USA).
    The activity of caspase-4 was evaluated after incubation with the molecules for 8 h according to the above-mentioned conditions for the MTT assay in 96-well black plates. Palmitic acid (1 mM) was used as positive control. After incubation, the supernatant was removed and caspase-4 substrate (50 µM) was added and further incubated for 150 min at 37 °C. Fluorescence was measured in a microplate reader (Cytation™ 3, BioTek, Winooski, VT, USA) at 400 nm/505 nm.