PST Analogue & Curcumin for Osteosarcoma Therapy Propidium Iodide (PI) Staining PI (Sigma-Aldrich Canada, Mississauga, ON, Canada) was used to detect cell lysis. Cells were incubated with 1 mg/mL PI for 10 minutes and images were taken at 4006 magnification on a Leica DM IRB inverted fluorescence microscope (Wetzlar, Germany). Results JCTH-4 causes selective cytotoxicity in OS cells in a time and dose-dependent manner As we have previously found PST (Figure 1A) to be effective in producing cytotoxicity selectively in various cancer cell lines, we evaluated the activity of JCTH-4 (Figure 1B) in the OS cell lines Saos-2 and U-2 OS using the WST-1 based colorimetric assay for cell viability. JCTH-4 decreased cell viability in a time and dose dependent manner and had a half-maximal inhibitory concentration (IC50) value of 0.25 mM after 48 hours in both Saos-2 and U-2 OS cells (Figure 2A,B). Importantly, JCTH-4 was selective to OS cells as HOb and NFF cells were drastically less sensitive to this compound (Figure 2C). Although HOb cells are the non-cancerous counterpart to the OS cell lines used in this study, we also evaluated the toxicity of this compound on another non-transformed non-cancerous human fetal fibroblast cell line (NFF) to confirm the selective anti-cancer activity of this compound. Figure 2. JCTH-4 causes selective cytotoxicity in OS cells in a time and dose-dependent manner. Effect of JCTH-4 on cellular viability of OS cells was determined by the WST-1 based colorimetric assay. (A) Saos-2 and (B) U-2 OS cells were treated with JCTH-4 and the WST-1 reagent was used to quantify cell viability. Absorbance was read at 450 nm and expressed as a percent of the control (Me2SO). Values are expressed as mean 6 SD from quadruplicates of 3 independent experiments. *p,0.05, **p,0.01, ***p,0.001 versus solvent control (Me2SO). (C) Effect on cellular viability of HOb and NFF cells treated with JCTH-4 compared to Saos-2 and U-2 OS cells after 72 hours. The WST-1 reagent was used to quantify cellular viability. Absorbance was read at 450 nm and expressed as a percent of the solvent control (Me2SO). Values are expressed as mean 6 SD from quadruplicates of 3 independent experiments. *p,0.005 versus Saos-2 cells; #p,0.005 versus U-2 OS cells. doi:10.1371/journal.pone.0028780.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28780