Acridine orange

Two lines of human osteosarcoma cells were used in this study. U-2 OS cells were obtained from Dr. Sheau-Yann Shieh (Institute of Biomedical Sciences, Sinica Academia, Taipei, Taiwan) and MG-63 cells were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Both lines of cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/mL of penicillin, and 100 g/mL of streptomycin (Gibco). Zoledronic acid and cisplatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Ursolic acid, N-acetyl-l-cysteine, acridine orange, and 3-methyladenine were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Reagents used were as follows: letrozole (Sigma, L6545), 17β-estradiol (E2) (Sigma, 491187), celecoxib (Sigma, PZ0008), PGE2 (Sigma, P5640), tunicamycin (Sigma, T7765), salubrinal (Calbiochem, 324895), rapamycin (Sigma, R0395), arachidonic acid (Sigma, A9673), MTT (Sigma, M2128), Z-VAD-fmk (Sigma, V116), bafilomycin A1 (Sigma, B1793), 3-MA (Sigma, 08592), Hoechst 33342 (Sigma, B2261), acridine orange (Sigma, A6014), DMSO (Sigma, D2650). letrozole, E2, celecoxib, PGE2, tunicamycin, and salubrinal were dissolved in DMSO, while MTT, Hoechst 33342, and acridine orange were dissolved in phosphate-buffered saline (PBS).
Antibodies were obtained from the following sources: antibodies against Beclin 1, COX-2, EP-4, β-actin, Bcl-2, BAX, phospho-4EBP1 and phospho-Akt (S473) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Phospho-p70-S6K(T389), eIF2α, phospho-eIF2α, Raptor, phospho-mTOR(S2448), caspase 3, and phospho-S6(S235/236) were from Cell Signaling Inc (Beverly, MA); LC3, ATG5, protein disulfide isomerase (PDI) were from Abcam (Cambridge, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Acridine orange (Santa Cruz Biotechnology, CA, USA) and propidium iodide (Santa Cruz Biotechnology, CA, USA) (AO/PI) fluorescent dyes were used to test the microscopic morphology and changes of HCT116 cells following treatment with 5-FU, diosmetin, and a combination of both. Cells were treated for 72 h with the IC₅₀ of monotherapy and combination therapy. Then, 10 μL of AO/PI mixture was used to stain the cell pellet. The morphological alterations in the cells were detected using a fluorescent inverted microscope. The resulting green, orange, and red colors represent viable, late apoptotic, and dead cells, respectively [13 (link)].