Winmdi 2

CM-H₂DCF-DA, dihydroethidine (DHE) and Mito-SOX fluorescent probes were used to measure the intracellular generation of hydrogen peroxide (H₂O₂), superoxide anions (O^2·−) and mitochondrial superoxide, respectively. Briefly, 3 × 10⁵ MDA-MB 435 cells were plated in 6-well plates and allowed to attach overnight. Cells were incubated with or without CDDO-Me and then incubated with 5 μM of H₂DCF-DA for 30 min, 20 μM of DHE for 30 min, or 2.5 μM MitoSOX-Red for 20 min in the dark at 37°C. After washing with Hank's Buffered Salt Solution (HBSS) containing Ca²⁺ and Mg²⁺, cells were further processed for fluorescence-activated cell sorting (FACS) analysis using a FACScan system (BD Biosciences). Data were analyzed using WinMDI 2.8 software (BD Biosciences).

Annexin FITC-conjugated (1 : 500) and propidium iodide (PI) fluorochrome-labeled cells were used to determine the nature of cell death (apoptosis or necrosis). After platting MCF-7 and MDA-MB-231 cells (3.5 × 10⁵ cells/mL) in a 12-well plates, they were incubated with AVME at 10 and 20 µM for MDA-MB-231 cells and 11 and 22 µM for MCF-7, or solvent control (DMSO) for 24 h. Cells were then washed twice with cold PBS and suspended again in a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂. The pellets were stained on ice in the dark for 15 min with a fluorescent probe solution containing 50 mg/mL PI and 1 mg/mL Annexin. The total percentage of cells experiencing apoptosis was demarcated as the sum of both early and late facets of apoptosis (Annexin V-FITC positive), lower and upper right quadrants in the two-parameter dot plots, as previously described by Robles-Escajeda et al. [26 (link)]. The WinMDI 2.9 software and the flow cytometer BD FACSVerse (Becton Dickinson, Franklin Lakes, NJ, USA) were used for analysis. Each experiment was repeated thrice.

The dead, necrotic, and apoptotic cells were analyzed by annexin V/PI double staining method, which was used to detect the externalization of phosphatidylserine (PS). Briefly, cells at the density of 4.5 × 10⁴ cells/well were seeded into 6-well plates and treated with or without DKPs for 24 h. Subsequently, both floating and adherent cells were harvested by centrifugation at 1,500 rpm for 5 min, then re-suspended in 300 μL of 1X binding buffer, followed by 3 μL of annexin V-FITC and propidium iodide (PI) (Apoptosis Detection kit, Lead Gen, Taiwan) was added into each sample and incubated for 20 min at room temperature in the dark following the protocol recommended by the manufactures. Cells were analyzed by flow cytometry (FACS Calibur, Becton Dickinson, CA, USA) using WinMDI 2.9 software (La Jolla, CA, USA). The individual populations can be defined using quadrant gates, the number of cells in each quadrant indicated the following: Quadrant-1 (Q1): annexin V^-/PI⁺ cells were considered as dead cells; Quadrant-2 (Q2): Annexin V⁺/PI⁺ cells were considered as late apoptotic and necrotic cells; Quadrant-3 (Q3): Annexin V⁺/PI⁻ cells were considered as early apoptotic cells; Quadrant-4 (Q4): Annexin V^-/PI^- cells were considered as healthy. Data were analyzed using FlowJo V12.1 software (Tree stat, Stanford, CA, USA).

Prior to analysis, serum was deprived for 24 h to synchronize cell cycle. Serum was then added back into the culture medium containing various doses of cantharidin (0, 2.5, 5, and 10 μM). After the treatments with cantharidin for 24 hours, MDA-MB-231 cells were fixed with 80% cold ethanol, and incubated with 0.5% Triton X-100 solution containing 1 mg/mL RNase A at 37°C for 30 min. Afterwards, propidium iodide (PI, Sigma) was added into each well at a final concentration of 50 μg/mL and the cells were further incubated for 30 min in the dark. Cell cycle progression was analyzed by a FACS (Becton Dickinson, USA). Data were processed using WinMDI29 software (Becton Dickinson).