Fluoxetine

(A) Dose-dependent effects of IR on the viability of RCC cells. Cells were treated with 0, 2, 4 or 6 Gy IR for 24 h. *P<0.05, IR vs. control. (B) Time-dependent effects of IR on the viability of RCC cells. Cells were treated for 0, 12, 24 or 36 h with 4 Gy IR. *P<0.05, IR vs. control. (C) Lactate dehydrogenase cytotoxicity assay was performed at different times and different doses of IR treatment in OS-RC-2 cells. *P<0.05 vs. untreated cells. (D) Cytotoxic effects in 786-O cells treated with IR (4 Gy) and/or ubenimex (0.5 mg/ml). The IR+Akt inhibitor and IR+ubenimex+Akt agonist were also tested. *P<0.05 vs. control. #P<0.05, IR+ubenimex or IR+Akt inhibitor vs. IR treatment. &P<0.05, IR+ubenimex+Akt agonist vs. IR+ubenimex treatment. (E) Radiation dose-response survival curves of 786-O cells with or without ubenimex. All results are presented as the mean ± standard deviation from three independent experiments. IR, irradiation; RCC, renal cell carcinoma.

Acridine orange-ethidium bromide staining in OS-RC-2 cells treated with IR (4 Gy) or ubenimex (0.5 mg/ml) alone or in combination for 24 h. Red fluorescence was used to identify autophagic cell death (). (Aa) Control; (Ab) Cells treated with ubenimex (0.5 mg/ml) alone; (Ac) Cells treated with IR (4 Gy) alone; (Ad) cells treated with combined ubenimex (0.5 mg/ml) and IR (4 Gy). Significant cell death was observed for the combined treatment of ubenimex and IR compared with ubenimex or IR alone. The percentage of positively stained cells was calculated and considered to be the death rate (%), which is the number of cells undergoing programmed cell death per 100 cells. *P<0.05, vs. control. All data are presented as the mean ± standard deviation from three independent experiments. IR, irradiation.

(A) OS-RC-2 cells were treated with IR (4 Gy) or ubenimex (0.5 mg/ml) alone or in combination for 24 h, and then detected using flow cytometry. (B) Western blotting for microtubule-associated proteins 1A/1B light chain 3B in OS-RC-2 cells. Cells were treated with IR (4 Gy) or ubenimex alone or in combination for 24 h. Actin was used as a loading control. Bars indicate the mean ± SD from three independent experiments (*P<0.05). (C) Early apoptosis, detected using an annexin V apoptosis detection kit, was measured using flow cytometry. Cells were treated with IR (4 Gy) and/or ubenimex (0.5 mg/ml). *P<0.05 vs. control. (D) Autophagic cell death. Fluorescein isothiocyanate-stained cells treated with IR (4 Gy) or ubenimex (0.5 mg/ml) alone or in combination for 24 h were detected using flow cytometry. *P<0.05 vs. control. Data are presented as the mean ± SD of three independent experiments. SD, standard deviation; IR, irradiation; FITC, fluorescein isothiocyanate; PI, propidium iodide; LC3B, microtubule-associated proteins 1A/1B light chain 3B.

(A) Electron microscopy microphotographs of OS-RC-2 cells treated with IR (4 Gy) or ubenimex (0.5 mg/ml) alone or in combination for 24 h. (Aa) Control; (Ab) Cells treated with ubenimex (0.5 mg/ml) alone; (Ac) Cells treated with IR (4 Gy) alone; (Ad) cells treated with combined ubenimex (0.5 mg/ml) and IR (4 Gy) The red arrows indicate autophagic vacuoles and autolysosomes. Untreated cells were used as a control. (B) Quantification of autophagosomes in 12 random fields in three independent experiments. *P<0.05 vs. control. (C) Western blotting for Akt and p-Akt in OS-RC-2 cells. Cells were treated with IR (4 Gy) or ubenimex (0.5 mg/ml) alone or in combination for 24 h. Actin served as a loading control. Bars indicate the mean ± standard deviation from three independent experiments (*P<0.05). IR, irradiation; p-, phosphorylated.