IR dose–response survival curves and cytotoxic effects resulting from ATO and IR in LNCaP and PC-3 cells.

<p>(A) Time-course and dose-dependent effects of IR on the viability of LNCaP and PC-3 cells. Cells were treated with 2, 4, 6 or 8 Gy of IR for 6, 12, 18, 24 and 48 hrs. (B) Time-course and concentration-dependent effects of ATO on the viability of LNCaP and PC-3 cells. Cells were treated with 2, 5, 10 or 15 µM of ATO for 12, 24, 36 and 48 hrs. (C) Cytotoxic effects of cells treated with IR (4 Gy) and ATO (5 µM). (D) The radiation dose–response survival curves of LNCaP and PC-3 cells with or without ATO. Data are presented as the mean ± standard deviation from three independent experiments.</p

Tumor growth and body weight of tumor-bearing mice treated with IR (6 Gy) or ATO (6 mg/kg×6) alone or in combination.

<p>(A) Measurement of body weight in nude mice once per week. (B) Measurement of tumor volume in nude mice every two days. Data are presented as the relative tumor volume (mean ± standard error) normalized to the initial tumor volume measured on day 0. (C) Direct observations of mice with tumors. After the experiments, the mice were sacrificed, and the tumors were removed. (D) Measurement of tumor weight in the nude mice after sacrifice. (E) Immunohistochemical (IHC) staining of PC-3 mouse xenograft tissues. IHC was used to determine the expression levels of PCNA, LC3 and Atg5 (×200 objective magnification).</p

Measurement of autophagy, apoptosis and cytotoxic effects in LNCaP and PC-3 cells pre-treated with 3-MA.

<p>(A)(D) Effects of 3-MA on cytotoxicity induced by combined treatment. (B)(E) Early apoptosis was measured by flow cytometry with Annexin V. (C)(F) Quantification of AVOs with AO using flow cytometry. *, <i>p</i><0.05, ATO+IR versus ATO+IR+3-MA.</p

Comparison of tumor growth inhibition, tumor volume quadrupling time, and tumor growth delay time of PC-3 tumors in nude mice.

<p>*Tumor growth inhibition rate was calculated based on the tumor volume on Day 18.</p>#<p><i>p</i> values for comparison of tumor growth delay time.</p

Measurement of autophagy in LNCaP and PC-3 cells that received various treatments.

<p>(A) Microphotograph of AVOs in LNCaP and PC-3 cells. Detection of green and red fluorescence in acridine orange (AO)-stained cells was performed using a fluorescence microscope. The <i>white arrows</i> point to AVOs. (B) EM microphotographs of PC-3 cells treated with IR (4 Gy) and ATO (5 µM) for 48 hrs. The <i>white arrows</i> point to autophagic vacuoles and autolysosomes. (C) Development of AVOs in LNCaP and PC-3 cells. Detection of green and red fluorescence in AO-stained cells using flow cytometry. (D) Quantification of AVOs with AO-stained cells treated with IR (4 Gy) or ATO (5 µM) alone or in combination using flow cytometry. Data are presented as the mean ± standard deviation from three independent experiments. #, <i>p</i><0.05, IR versus combined treatment. *, <i>p</i><0.05, ATO versus combined treatment. (E) (F) Western blotting of LC3-I, LC3-II, p62/SQSTM1, Atg5 and Atg5-12 expression in LNCaP and PC-3 cells. Cells were treated with IR (4 Gy) and ATO (5 µM) for 48 hrs.</p

Effects of Akt/mTOR signaling pathway protein expression in LNCaP and PC-3 cells treated with IR and ATO alone or in combination.

<p>Cells were treated with IR (4 Gy) and ATO (5 µM) for 48 hrs.</p

Measurement of apoptosis in LNCaP and PC-3 cells that received various treatments.

<p>(A) Early apoptosis detection was measured by flow cytometry with an Annexin V apoptosis detection kit. Cells were treated with IR (4 Gy) and ATO (5 µM) for 48 hrs. #, <i>p</i><0.05, IR versus combined treatment. *, <i>p</i><0.05, ATO versus combined treatment. (B) (C) ROS generation in PC-3 cells treated with 5 µM ATO or 4 Gy IR alone or in combination for 30, 60, 120 min and with DCFH-DA for an additional 30 min. The fluorescence in the cells was immediately assayed using flow cytometry. #, <i>p</i><0.05, IR versus combined treatment. *, <i>p</i><0.05, ATO versus combined treatment. (D) Western blotting of PARP, cleaved-PARP, pro-caspase 3, cleaved-caspase 3and Bcl-2. The level of total GAPDH protein was used as the loading control. Cells were treated with IR (4 Gy) and ATO (5 µM) for 48 hrs. Data are presented as the mean ± standard deviation from three independent experiments.</p

Comet assay and ER stress induced by IR and/or MP in PC-3 cells. (A) EtBr staining of cells treated with IR (4

<p> <b>Gy) and MP (25 </b><b>μM).</b> The tails indicate DNA damage. (B) The effect of MP or IR alone or in combination for 48 hrs on average tail DNA. #, <i>p</i><0.05, IR versus combined treatment. *, <i>p</i><0.05, MP versus combined treatment.</p

Comparison of tumor growth inhibition, tumor volume quadrupling time, and tumor growth delay time of PC-3 tumors in nude mice.

*<p> <b>Tumor growth inhibition rate was calculated based on the tumor volume on ay 10.</b></p><p> <b># </b><b><i>p</i></b><b> values for comparison of tumor growth delay time.</b></p

IR dose–response survival curves and cytotoxic effects resulting from MP and/or IR in PC-3 cells.

<p>(A) Chemical structure of MP. (B) Concentration-dependent effects of MP on the viability of PC-3 cells. Cells were treated with 5, 15, 25, 35 or 45 μM MP for 48 hrs. *, <i>p</i><0.05, MP versus control. (C) Cytotoxic effects in cells treated with IR (4 Gy) and/or MP (25 μM). #, <i>p</i><0.05, IR versus combined treatment. *, <i>p</i><0.05, MP versus combined treatment. (D) The radiation dose-response survival curves of PC-3 cells with or without MP. Data are presented as the mean ± standard deviation of three independent experiments.</p