Effect of Daiokanzoto on <i>P</i>. <i>gingivalis</i>-induced activation of the NF-κB signaling pathway in monocytes.

<p>Data are expressed as means ± standard deviations. *, Significant inhibition at <i>p</i> < 0.01 using a Student’s <i>t</i>-test.</p

Effect of Daiokanzoto on IL-6 (Panel A) and CXCL8 (Panel B) secretion by LPS-stimulated human gingival fibroblasts (HGF-1).

<p>Data are expressed as means ± standard deviations. *, Significant inhibition at <i>p</i> < 0.01 using a Student’s <i>t</i>-test.</p

Effect of Daiokanzoto on IL-6 (Panel A) and CXCL8 (Panel B) secretion by LPS-stimulated human oral epithelial cells (OBA-9).

<p>Data are expressed as means ± standard deviations. *, Significant inhibition at <i>p</i> < 0.01 using a Student’s <i>t</i>-test.</p

Effect of Daiokanzoto on the expression of <i>fimA</i> and <i>hagA</i> (Panel A) and <i>rgpA</i> and <i>rgpB</i> mRNA (Panel B) in <i>P</i>. <i>gingivalis</i>.

<p>Data are expressed as means ± standard deviations. mRNA expression was normalized to 16S rRNA. *, significantly different (P < 0.01) compared to an untreated control.</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 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

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

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

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