TJ-84 attenuated 5-FU-induced NO production.

<p>(A), Sa3 cells were incubated with or without 5 mg/mL of 5-FU for 3 h following a 1-h pre-incubation with 500 µg/mL of TJ-84. DAF-2DA was then loaded for 30 min. The production of NO (green) were detected by fluorescence microscopy. (B), The fluorescence intensity per cell was calculated using ImageJ. The calculation of the red/green ratio is shown on the graph. Values are means ± S.E.M. (n = 50). **<i>p</i><0.01 compared to the control cells.</p

Hypothetical model of how TJ-84 decreases 5-FU-induced Sa3 cell death.

<p>TJ-84 decreased 5-FU-induced cell death by inhibiting ROS production. See text for details.</p

TJ-84 suppressed 5-FU-induced ROS production in mitochondria.

<p>(A), Sa3 cells were incubated with or without 5 mg/mL of 5-FU for 6 h following a 1-h pre-incubation with 500 µg/mL of TJ-84. MitoSOX Red (5 µM) and 100 µM Mitotracker Green were then loaded for 30 min. ROS (red) and mitochondria (green) were detected by fluorescence microscopy. (B), The red fluorescence intensity per cell was calculated using ImageJ and is shown on the graph. Values are means ± S.E.M. (n = 63, 71, 75). **<i>p</i><0.01 compared to the control cells.</p

TJ-84 attenuated 5-FU-induced mitochondrial depolarization.

<p>(A), Sa3 cells were incubated with or without 5 mg/mL of 5-FU for 3 h following a 1-h pre-incubation with 500 µg/mL of TJ-84. JC-1 (1 µg/mL) was then loaded for 30 min. JC-1 aggregates (red) and monomers (green) were detected by fluorescence microscopy. (B), The fluorescence intensity per cell was calculated using ImageJ. The calculation of the red/green ratio is shown on the graph. Values are means ± S.E.M. (n = 20, 16, 14). **<i>p</i><0.01, *<i>p</i><0.05 compared to the control cells.</p

TJ-84 reduced 5-FU-induced cell death.

<p>(A), Cytotoxicity of TJ-84. Sa3 cells were incubated with various concentrations of TJ-84 for 24 h, and cell viability was then measured using WST-8 kits. Values are means ± S.E.M. (n = 3). **<i>p</i><0.01 compared to control cells that had not been incubated with TJ-84. (B), Viability of cells incubated with various concentrations of TJ-84 for 1 h and then incubated with 5-FU for 24 h. Results are expressed as percentages with respect to control cells that had not been incubated with TJ-84 and 5-FU. Values are means ± S.E.M. (n = 4). **<i>p</i><0.01 compared to control cells. (C), The effect of TJ-84 on LDH release from cells incubated with 5-FU for 24 h was assessed using WST-8 kits. The supernatant of Sa3 cells incubated with 0.1% Triton-X for 5 min was used as a positive control (Triton). The LDH levels in the supernatants are expressed as percentages with respect to cells that had not been incubated with 5-FU. Values are means ± S.E.M. (n = 4). *<i>p</i><0.05 compared to the control cells.</p

Inhibition of NLRP3 inflammasomes decreased 5-FU-induced cell death.

<p>(A), Cell viability of Sa3 cells incubated with 5 mg/mL of 5-FU for 24 h after a 30-min pre-incubation with caspase inhibitor at each concentration. Values are means ± S.E.M. (n = 4). **<i>p</i><0.01 compared to the control group. (B), Expression of NLRP3 mRNA in Sa3 cells treated with NLRP3 siRNA (NLRP3) or scrambled oligo (scr). Values are means ± S.E.M. (n = 4). *<i>p</i><0.05 compared to cells without oligo (–). (C), Cell viability of cells transduced without (–) or with NLRP3 siRNA (NLRP3) or control oligo (scr) following an incubation with (closed bar) or without (open bar) 5 mg/mL of 5-FU for 3 h. Data are given as percentages compared to the group that was not incubated with 5-FU. Values are means ± S.E.M. (n = 6). *<i>p</i><0.05 compared to the control group. (D), Effect of transfection with NLRP3 siRNA (NLRP3) or scrambled oligo (scr) on 5-FU-induced LDH release. The LDH levels in the supernatants are given as percentages of cells not incubated with 5-FU and not transduced with siRNA. Values are means ± S.E.M. (n = 8). *<i>p</i><0.05 compared to the control cells.</p

NF-κB did not attenuate 5-FU-induced cell death.

<p>(A), Sa3 cells were incubated with or without 5 mg/mL of 5-FU for 1 h and 3 h. The cells then stained with anti-p65 antibody and the localization of p65 was detected by florescence microscopy. (B), Cell viability of Sa3 cells incubated with 5 mg/mL of 5-FU for 24 h after a 30-min pre-incubation with NF-κB inhibitors, 5 µM BAY or 200 µM CAPE. Values are means ± S.E.M. (n = 4). **<i>p</i><0.01 compared to the control group.</p

5-FU-activated inflammasome pathway.

<p>Sa3 cells were incubated with 5 mg/mL of 5-FU for 0 to 24 h. (A), Western blot analysis of the expression of NLRP3 and the precursor of caspase-1 (pro-casp-1) in cell lysates. (B), Western blot analysis of cleaved caspase-1 (p20) in supernatants. Arrow indicates p20-specific bands. (C), ELISA assay of IL-1β in supernatants of Sa3 cells incubated without (open box) or with (closed box) 5 mg/mL of 5-FU for 6 h and 24 h. Values are means ± S.E.M. (n = 4). **<i>p</i><0.01 compared to Sa3 cells incubated without 5-FU.</p

Tumor morphologies and metastatic potentials of the prostate-derived cell lines.

<p>Tumor morphologies and metastatic potentials of the prostate-derived cell lines.</p

Enlargement of GLA due to slow cell proliferation and inter-gla fusion in the 3d stemness-inducing nanoenvironment.

<p>(A) Representative photomicrographs of PC-3 cells after reaching confluent. Cells were cultured in the 2D condition. Cellular morphologies at day 4, 5, and 7 were shown. Arrowheads indicate GLA on the 2D monolayer cells. Scale bar, 100 μm. (B) Representative photomicrographs of PC-3 cells in the 3D culture condition. Cellular morphologies at day 11 and 14 were shown. Scale bar, 100 μm. (C) Growth curves of PC-3 cells cultured in 2D serum-contained and 3D stem cell medium conditions. Cells were cultured in a 96-well plate. **P < 0.01 (2D serum vs 3D stem), n = 3. (D) Growth curves of PC-3 cells cultured in 2D serum-contained and 2D stem cell medium conditions. *P < 0.05 (2D stem vs 2D serum), n = 3. (E) Growth curves of PC-3 cells cultured in 3D serum-contained and 3D stem cell medium conditions. *P < 0.05 (3D stem vs 3D serum), n = 3. **P < 0.01 (3D stem vs 3D serum), n = 3. (F-H) Viabilities of PC-3 cells cultured in 2D or 3D conditions in serum-contained or stem cell media. Same data with different vertical axis values were shown between F and H and between G and I. (F, H) P < 0.05 (2D serum vs 2D stem), n = 3. (G, I) *P < 0.05 (vs day 0), n = 3. **P < 0.01 (vs day 0), n = 3. 3D serum d0 vs d7, P = 0.028. 3D serum d0 vs d11, P = 0.0052. 3D serum d0 vs d14, P = 0.0012. 3D stem d0 vs d7, P = 0.0138. 3D stem d0 vs d11, P = 0.0007. 3D stem d0 vs d14, P = 0.0004.</p