Fig. 2
The drug combination enriched iROS production and promoted cytotoxicity compared to Sal or Das alone, in a time-dependent manner. a Plots of the drug-induced iROS level versus time. The induction of iROS by the drug combination followed a 2-step kinetic. MDA-MB-468, MDA-MB-231, and MCF-7 cell lines were treated with individual drugs (at the corresponding IC50 concentration (Fig. 1c)) or the 2-drug mixture prior to incubation with DCF-DA for FACS analysis of the iROS level. The mean fluorescence was calculated by comparison with PBS-treated cells (control). b Fluorescence microscopy confirmed that the drug-induced iROS increase in treated MDA-MB-468 cells was time-dependent. Prior to imaging, the cells were treated with DCF-DA and DAPI for staining the iROS (green) and nucleus (blue), respectively. Scale bar is 45 μm. c A flow cytometry graph showing the increase of the apoptotic (AnV+PI−)/dead (AnV+PI+) cell population in response to the drug treatments. MDA-MB-468 cells were incubated with Sal (0.5 μM), Das (15 μM) or the 2-drugs combination for 72 h prior to staining with AnV-FITC and PI for FACS analysis. d Graph bars showing the percentage of healthy, apoptotic, necrotic, and dead cells following treatment with PBS (control) Sal, Das, or S + D for 72 h (see Fig.S6 for detailed quantification of the cell populations). e Representative microscopic images of MDA-MB-468 cells 72 h after drug incubation. Scale bar is 25 μm. f Cell viability assay showed that the cytotoxicities of Sal and Das alone or the drug combination were also time-dependent. g-h Comparing the cytotoxicities of the same drug combination applied sequentially and concurrently for treating MDA-MB-468 cell line. The cells were treated sequentially with Sal and followed by Das (Sal--Das) or Das and then Sal (Das--Sal), or concurrently with Sal and Das (S + D). The concentration of Sal and Das used in this study was 0.5 and 15 μM, respectively. Plots showing the changes in the (g) iROS level and (h) ratio of the drug-treated to non-treated cell numbers over time. All the experiments were independently performed in triplicate