Caspase 9 activity

24R,25(OH)₂D₃ was purchased from Enzo Life Sciences (Farmingdale, NY, USA, BML-DM300) to assess the effect of 24R,25(OH)₂D₃ on markers of tumorigenicity. UM-SCC-12 and UM-SCC-11A cell monolayers were first cultured on 96-well or 24-well plates (n = 6 wells/group). At the time of treatment, the cells were treated with 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M 24R,25(OH)₂D₃, or vehicle for 15 min, at which point the media were replaced with fresh full media for 24 h. The cells were then assessed for proliferation via the EdU DNA synthesis assay (Thermo Fisher Scientific), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) (Sigma-Aldrich, St. Louis, MO, USA). Apoptosis was measured via the p53 assay (R&D Systems, Minneapolis, MN, USA), DNA fragmentation (TUNEL assay) (Trevigen, Gaithersburg, MD, USA), BAX/BCL2 mRNA, and Caspase 9 Activity (Promega, Madison, WI, USA). Metastasis was measured through the Western blot analysis of Nidogen-1 and the ratio of osteoprotegerin to RANKL, two lung and bone metastasis markers, respectively.