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

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

Measurement of degradation of collagen by MMPs in NIH 3T3 cells treated with UVB and/or FIR.

<p>(A) MMP-1 and MMP-9 expression in UVB-irradiated NIH 3T3 cells. Cells were irradiated with 40, 80 or 120 mJ/cm² UVB for 48 h. (B) The expression levels of MMP-1 and MMP-9 proteins were measured by western blot analysis following treatment with UVB and/or FIR. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (C) Levels of collagen type 1 in the culture medium were measured by ELISA. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. *p<0.05, UVB versus UVB+FIR. The data are presented as the means ± standard deviation of three independent experiments.</p

The protein expression and density of collagen fibers in a UVB-exposed hairless mouse model.

<p>(A) Masson’s trichrome staining estimated for relative collagen density. IHC staining of skin tissues was used to determine the expression levels of MMP-9 (B) and LC3 (C). (D) Western blot analysis of protein expression in skin tissues.</p

FIR pathways and effects in skin photoaging.

<p>FIR inhibits MMPs and leads to interference with collagen degradation. Furthermore, FIR increases collagen synthesis through the TGF-β/Smad pathway. In addition, FIR-induced autophagy may be mediated by inhibition of the Akt/mTOR signaling pathway. Autophagy can block the epidermal hyperproliferative response to UV and may suppress photoaging.</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 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

Measurement of autophagy and the Akt/mTOR signaling pathway in NIH 3T3 cells treated with UVB and/or FIR.

<p>(A) Immunofluorescence staining of LC3 protein in NIH3T3 cells treated with UVB and/or FIR. Representative cell images showing punctate LC3 distribution using a confocal microscope. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (B) Quantitative data calculating the percentage of LC3-positive cells. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. *, p<0.05, versus control. The data are presented as the means ± standard deviation of three independent experiments. (C) The expression levels of autophagic-related proteins were measured by western blot analysis following treatment with UVB and FIR alone or in combination. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 24 h. (D) The expression levels of Akt/mTOR signaling-associated proteins were measured by western blot analysis. Cells were irradiated with 80 mJ/cm² UVB for 24 h. Then, the cells were treated with FIR for 30 min and cultured for 6 h.</p