PST Analogue & Curcumin for Osteosarcoma Therapy Figure 7. JCTH-4 alone and with CC does not induce phosphatidylserine externalization in HOb and NFF cells. Annexin V binding to externalized phosphatidylserine was monitored to confirm that apoptosis was not induced in (A) HOb and (B) NFF cells after 72 hours of treatment. Cells were treated with the indicated concentrations of JCTH-4, CC and solvent control (Me2SO). Cells were also stained with Hoechst dye. Images were taken at 4006 magnification on a fluorescent microscope. Green fluorescence is indicative of Annexin V binding to externalized phosphatidylserine of the plasma membrane. Scale bar = 15 mm. All images are representative of 3 independent experiments. doi:10.1371/journal.pone.0028780.g007 Figure 8. JCTH-4 dissipates MMP alone and in combination with CC in OS cells. Effect of JCTH-4 and CC on MMP in (A) Saos-2 cells after 96 hours of treatment and (B) U-2 OS cells after 72 hours of treatment was examined by TMRM staining. Cells were grown on coverslips, treated with the indicated concentrations of JCTH-4, CC, and solvent control (Me2SO) and stained with TMRM and Hoechst dye. Images were taken at 4006 magnification on a fluorescent microscope. Red fluorescent punctuate marks are indicative of mitochondria with intact MMP. Scale bar = 15 mm. All images are representative of 3 independent experiments. doi:10.1371/journal.pone.0028780.g008 PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28780