Effects of EGCG on NO-induced caspase-1 activation <i>in vitro</i> and <i>ex vivo.</i>

<p>Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 24 h. (A) IL-1β concentrations were measured in cell supernatants using the ELISA method. (B) Protein extracts were assayed for cleaved caspase-1 by western blot analysis. (C) Rat organ of Corti explants were pretreated with 50 μM EGCG for 2 h, followed by treatment with 500 μM SNAP. After the explants were homogenized, caspase-1 levels were confirmed using a caspase-1 assay kit. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on NO-induced apoptosis-related genes in HEI-COΙ cells.

<p>(A) Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 24 h. After isolation of cytosolic and mitochondrial fractions, the protein extracts were assayed for cyt <i>c</i> by western blot analysis. GAPDH was used as an internal cytosolic marker control, and VDAC was used as a mitochondrial marker. (B) The relative levels of cytosolic and mitochondrial cyt <i>c</i> were quantified by densitometry. (C) Western blot analysis revealed a reduction in Bcl-2 protein after SNAP exposure. (D) Relative levels of Bcl-2 are shown. (E) The levels of cleaved caspase-3 after treatment with EGCG were assayed by western blot analysis. (F) The effect of EGCG on caspase-3 activation was determined using a colorimetric kit. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on NF-κB activation in the organ of Corti.

<p>Organ of Corti explants isolated from rats were pretreated with 50 μM EGCG for 2 h, followed by treatment with 500 μM SNAP for 4 h. Explants were fixed, stained with TRITC-conjugated phalloidin (NF-κB, red) and DAPI (nuclear, blue), and examined under an Olympus microscope (magnification, 100×).</p

Effects of EGCG on NO-induced MMP loss in HEI-OC1 cells.

<p>(A) MMP levels were measured by flow cytometry using the fluorescent probe DiOC₆. SNAP incubation (250–500 μM) resulted in a left shift of the cell distribution, indicating reduced MMP. (B) The mean fluorescence intensity of the traces is depicted in panel A. (C) Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 24 h. MMP levels were measured by flow cytometry. (D) The mean fluorescence intensity of the traces is depicted in panel C. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on NO-induced ROS production in HEI-OC1 cells.

<p>(A) ROS levels were measured using the fluorescent probe DCFH-DA and a spectrofluorometer. Cells were treated with 500 μM SNAP for varying times. (B) Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 1 h. The relative fluorescence levels were measured. (C) ROS production was measured by flow cytometry analysis. (D) The relative fluorescence levels of DAF-2/DA were measured. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on NO-induced NF-κB activation in HEI-COΙ cells.

<p>(A) After transfection, cells were treated with 500 μM SNAP for 4 h. After isolation of the nuclear fraction, protein extracts were assayed for NF-κB activity by western blot analysis. (B) Protein extracts were assayed for caspase-3 by western blot analysis. (C) Relative levels of NF-κB and caspase-3 are shown. (D) Cells were pretreated with 50 μM EGCG, followed by treatment with 500 μM SNAP for 4 h. After isolation of cytosolic and nuclear fractions, protein extracts were assayed for IκB-α and NF-κB by western blot analysis. (E) Cells were transfected with an NF-κB-dependent reporter gene for 48 h, and transfected cells were treated with SNAP for 4 h. EGCG (50 μM) was added 2 h prior to SNAP treatment. Cells were harvested, and luciferase activity was measured as described in the Materials and Methods. (F) Cells were fixed and stained with NF-κB (green) and DAPI (blue). Cells were then analyzed using an Olympus microscope (magnification, 100×). All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on organ of Corti explants.

<p>(A) Cochlear explants from the apical, middle, and basal turns of the rat cochlea were incubated with SNAP (250–500 μM) for 24 h. (B) Relative cell viability is shown. (C) Cochlear explants were incubated with SNP (300–500 μM) for 24 h. (D) Explants were treated with 200 μM L-NAME for 1 h and subsequently treated with 20 μM cisplatin. (E) Explants were treated with 10 μM C-PTIO for 1 h and subsequently treated with 500 μM SNAP. (F) Cochlea explants were treated with 50 μM EGCG and then 500 μM SNAP for 24 h. The explants were fixed with paraformaldehyde (4%), and TRITC-conjugated phalloidin (red), which binds to F-actin, was used to stain hair cells. (G) Relative cell viability is shown. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effects of EGCG on NO-induced cell death in HEI-OC1 cells.

<p>(A, B) Cell viability evaluated by MTT colorimetric assay, as a function of SNAP concentration and exposure time. (C) Cells were treated with 500 μM SNAP for varying times, and NO levels were determined by the measurement of nitrite based on the Griess reaction. (D) Cells were pretreated with 50 μM EGCG and then 500 μM SNAP for 24 h. Cell viability was evaluated by MTT assay. All data represent the mean ± SEM of 3 independent experiments (<i>^#P</i><0.05 vs. control, *<i>P</i><0.05 vs. SNAP alone).</p

Effect of β-lapachone on the cell viability of metastatic melanoma cells.

<p>(A and B) Cell viability of β-lapachone-treated B16F10 (A) and B16BL6 (B) cells. The cells were seeded at a density of 5 × 10³ cells/well in 96-well microplates and then treated with various concentrations of β-lapachone for 24–72 h. After incubation, cell viability was determined by WST assay. Values are the means ± SD of data from three independent experiments. (C) Morphology of β-lapachone-treated cells. B16F10 and B16BL6 cells were treated with β-lapachone for 48 h. The morphological changes of the cells were observed under a phase-contrast microscope (magnification 200×).</p

Effect of β-lapachone on the migration and invasion of B16F10 cells, through inhibition of MMP expression and activity.

<p>(A) Cancer cell motility was determined by wound healing assay. Cells were cultured to 8–90% confluent monolayer in a 6-well plate and scratched with a 200 μl micropipet tip. After washing with a serum-free medium, the cells were treated for 24 h with the indicated concentrations of β-lapachone. The denuded zone was observed at 0 and 24 h and images were photographed using a microscope (100× magnification). (B) Invasion ability of B16F10 cells was determined by invasion assay using matrigel-coated transwell chamber. (C) The mRNA expression levels of MMP-2 and MMP-9 were measured by real-time RT-PCR after β-lapachone treatment for 24 h. (D) Inhibitory effect of β-lapachone on activity of MMP-2 and MMP-9. After 12 h starvation, cells were treated with β-lapachone for 24 h and conditioned medium was collected for use in gelatin zymography. Photographs are representative of three independent experiments. The band intensity was quantitatively analyzed using ImageJ software. Values are the means ± SD of data from three independent experiments. *<i>P</i> < 0.05 and **<i>P</i> < 0.01.</p