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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Mol Cancer Res. 2015 Jan 30;13(5):819–827. doi: 10.1158/1541-7786.MCR-14-0492

Figure 4. Apoptosis assay to determine abrogation of TRAIL binding by OPG-mutants.

Figure 4

MDA-MB-435 cells were cultured in the presence of 100 ng of TRAIL and 200 ng of OPG-wt or OPG-mutants. As a positive control, MDA-MB-435 cells were cultured with TRAIL alone. After 24 hours, MDA-MB-435 cells were either fixed in 3.7% paraformaldehyde and then stained with 0.05% Crystal violet for 30 minutes and viewed using a light microscope (100x) (A) or cultured with 20 μl of the solution 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) for 2 hours and then measured at an absorbance of 490nm (B) (*p<0.05, **p<0.005, ***P<0.001, compared to control). To determine downstream activity of TRAIL function, a human osteolytic cancer cell line, PC3, was cultured in a combination of TRAIL and OPGwt or OPGmut (Y49R or F107A) for 3 hours. Cells lysates were prepared by harvesting the cultures and Western blot analysis was performed for cleaved caspase-3 activity (C).