Software summit version 6

To investigate cell death induced by vitamin C treatment, a flow cytometric analysis using the cell apoptosis kit Annexin V/PropidiumIodide (PI) double staining uptake (Life Technologies, Monza, Italy) was used. Control and PTC-derived cells, at the density of 5 × 10⁴ cells/mL, were seeded in 6-well plates (Corning, Tewksbury, MA, USA) with complete DMEM/F12. First, the cells were treated with 5 mM vitamin C for B-CPAP and K1, 10 mM for TPC-1 and 15 mM for NThy-ori3-1 for 48 h. Next, all cell lines were treated with 5 mM of vitamin C and with 10 mM of NAC, or with NAC and vitamin C together for 48 h. Cells were washed once with PBS 1X and stained, according to the kit’s protocol. Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter. For the following experiments, 5 mM of vitamin C was used in all cell lines.

To investigate the cell death induced by the treatment of compounds, a flow cytometric analysis was performed using the Annexin V-FITC/PI Apoptosis detection kit. Differentiated SH-SY5Y cells were plated in 6-well plates at the density of 3 × 10⁵ cell/well and were then treated with 125 and 250 μM of compounds for 24 h. After trypsinization, cells were washed once with PBS and re-suspended in 100 μL of Annexin binding buffer plus 5 μL of Annexin V fluorescein isothiocyanate and 1 μL of PI. After incubation in the dark for 15 min at room temperature, stained cells were analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using a 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter) (Brea, CA, USA). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter.

To assess which death mechanisms our compounds induced, cell apoptosis kit Annexin V/Propidium iodide (PI) double staining uptake (Invitrogen, Life Technologies, Italy) was used. 3b compound was selected as lead and employed in our assay. Human lung cancer cells (SK-MES 1) were seeded at the density of 8 × 10⁵ cells/ml in 6-well plates (Corning, United States) with a complete medium (described in cell culture section). After overnight incubations, the cells were treated with or without different concentrations of 3b for 96 h. Cells were then labeled with Annexin V and PI as previously described (Ibba et al., 2021 (link)). Stained cells were then analyzed by flow cytometry, measuring the fluorescence emission at 530 and 620 nm using 488 nm excitation laser (MoFloAstrios EQ, Beckman Coulter). Cell apoptosis was analyzed using Software Summit Version 6.3.1.1, Beckman Coulter.