Modfit lt ver 3

After treatment with the indicated concentrations of As^III and Tetra, alone or in combination, for 48 h, cell cycle analysis was performed using a FACSCanto flow cytometer (Becton–Dickinson, CA, USA) according to a method reported previously [12 (link), 34 (link)]. Briefly, cells were washed twice with PBS, fixed with 1% paraformaldehyde/PBS for 30 min, washed twice again with PBS, permeabilized in 70% (v/v) cold ethanol and kept at − 20 °C for at least 4 h. Cell pellets were then washed twice with PBS after centrifugation and incubated with 0.25% Triton-X 100 for 5 min on ice. After centrifugation and washing with PBS, cells were resuspended in 500 µl of PI/RNase A/PBS (5 µg/ml of PI and 0.1% RNase A in PBS) and incubated for 30 min in the dark at room temperature. A total of 10,000 events were acquired and Diva software and ModFit LT™ Ver.3.0 (Verity Software House, ME, USA) were used to calculate the number of cells at each G₀/G₁ and S phase fraction.

After treatment with 10 μM As^III+6.4 μM Tetra for 48 h, cell cycle analysis was performed using a FACSCanto flow cytometer (Becton–Dickinson) according to a method reported previously (Kikuchi et al., 2013 (link); Yao et al., 2017 (link)). Briefly, cells were washed twice with PBS, fixed with 1% paraformaldehyde/PBS for 30 min, washed twice again with PBS, permeabilized in 70% (v/v) cold ethanol and kept at −20°C for at least 4 h. Cell pellets were then washed twice with PBS after centrifugation and incubated with 0.25% Triton-X 100 for 5 min on ice. After centrifugation and washing with PBS, cells were resuspended in 500 μl of PI/RNase A/PBS (5 μg/ml of PI and 0.1% RNase A in PBS) and incubated for 30 min in the dark at room temperature. A total of 10,000 events were acquired and Diva software and Mod-Fit LT™ Ver.3.0 (Verity Software House, ME, USA) were used to calculate the number of cells at each G₀/G₁, S and G₂/M phase fraction. In order to explore whether JNK or autophagy contributes to the cytotoxicity of the combined regimen by modulating cell cycle progression, MDA-MB-231 cells were treated with JNK inhibitor or autophagy inhibitors at the indicated concentrations for 30 min prior to treatment with 10 μM As^III+6.4 μM Tetra in the presence or absence of each inhibitor for an additional 48 h, followed by the cell cycle analysis as described above.