Synergistic effect of treatment with TEM and CCM on apoptosis in renal cell carcinoma cell lines. (A) Caki-1 and OS-RC-2 cells were treated with TEM alone, CCM alone, TEM and CCM, or the Ctr for 48 h. Cells were stained using Annexin-V-Fluorescein and PI at room temperature for 20 min, and apoptosis was measured using flow cytometry. The percentage of apoptotic cells in the (B) Caki-1 and (C) OS-RC-2 cell lines was calculated and analyzed using GraphPad Prism software. Values are presented as the mean ± standard deviation from three independent samples (*P<0.05 vs. Ctr). Following treatment with TEM alone, CCM alone, TEM and CCM, or the Ctr for 48 h, DNA strand breakage was detected using the terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling assay and observed using a confocal laser scanning microscope in (D) Caki-1 cells and (E) OS-RC-2 cells. TEM, temsirolimus; CCM, curcumin; Ctr, control; PI, propidium iodide; dUTP-TRITC, 2′-deoxyuridine, 5′-triphosphate-tetramethylrhodamine; DAPI, 4′,6-diamidino-2-phenylindole.