Fig. 1

Sublethal cinobufagin increases ROS levels specifically in cancer cells. A Measurement of IC₅₀ by MTT. SW480 and SW1116 cancer cells were treated by 0.9, 2.7, 8.2, 24.7, 74.0, 222.2, 666.7 or 2000.0 nM CBG for 24, 48 or 72 h. IC₅₀ values were derived in the Prism 7 software. B Evaluation of cytotoxicity by MTT. Cells were treated by 100 nM CBG for 24 h. Cell viability was shown as the percentage of vehicle control of the corresponding cell line. Viability of the cancer but not noncancerous cell lines was reduced by 40–50%. C Representative images of cells stained by DCFH-DA. Cells were treated by 100 nM CBG for 3 h. Oxidization of DCFH-DA-derived, membrane-impermeable DCFH generated highly fluorescent 2’,7’-dichlorofluorescein (DCF). Intensity of DCF fluorescence served as a measure of cellular ROS. A prominent increase in ROS levels was induced in SW480 and SW1116 but not NCM460 cells, and NAC blocked the ROS increase in the cancer cells (scale bar: 25 μm). D, E Measurement of ROS levels by flow cytometry. Treatment by 100 nM CBG for 3 h caused a pronounced increase in ROS levels in SW480 and SW1116 but not NCM460 cells, NAC blocked the ROS increase in SW480 and SW1116 cells (D); significant increase in ROS levels in the cancer cells was evident 15 min after treatment by 100 nM CBG (E). F MTT assay. SW480 cancer cells were treated with 12.5, 25, 50 or 100 nM CBG, with or without NAC, for 24 h. NAC significantly reduced the cytotoxicity induced by CBG. n.s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs vehicle control or NAC-treated group (n = 3)