Possible mitochondrial molecular mechanism of BGP-15 cytoprotective action.

<p>BGP-15 reduces mitochondrial ROS production at complex I and at complex III, and so reduces ROS induced mitochondrial damage, as well as cell death.</p

BGP-15 attenuates hydrogen peroxide-induced mitochondrial reactive oxygen species production in WRL-68 cells.

<p>(A) Effect of hydrogen peroxide and BGP-15 pretreatment (for 30 minutes) on mitochondrial ROS production, as determined by the oxidation of the mitochondrial enriched dye from DHR123 to R123 in WRL-68 cells that had been labelled with mitochondrial directed red fluorescent protein. High magnification fluorescent microscopic images show the different localization of the produced R123. Inserts are expanded from the area indicated by dashed rectangles. (B) Quantification of R123 production. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 and ***P < 0.001 compared to control cells; ^##P < 0.01 compared to H₂O₂-treated cells.</p

BGP-15 attenuates lipopolysaccharide-induced mitochondrial depolarization and production of reactive oxygen species.

<p>(A) Effect of BGP-15 on LPS-induced mitochondrial membrane depolarization in U-251 MG cells. Cells were exposed to 1 μg/mL LPS in the absence or presence of 50 μM BGP-15 for 1 hour, then stained with 100 ng/mL of JC-1. Fluorescent microscopic images were taken using both the red and green channels. Representative merged images of three independent experiments are presented. (B) Quantitative analysis of LPS-induced (1 μg/mL) mitochondrial depolarization and its reduction by BGP-15 (50 μM) in U-251 MG cells. Results are presented as the mean ± SEM. **P < 0.01 and ***P < 0.001 compared to control cells; ^#P < 0.05 compared to LPS-treated cells. (C) Effect of BGP-15 on LPS-induced mitochondrial membrane depolarization in U-251 MG cells. Cells were treated with 1 μg/mL LPS in the absence or presence of 50 μM BGP-15 for 1 hour, then stained with 50 nM of TMRM. After a 15 minutes incubation fluorescent signal was measured by the GloMax Multi Detection System, then remeasured after the application of 1 μM FCCP. ΔΨ was calculated as the difference of fluoresescence signal before and after FCCP-treatment. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 compared to control cells; ^##P < 0.01 compared to LPS-treated cells. (D) Effect of BGP-15 on the LPS-induced ROS production in U-251 MG cells (containing the TLR4 receptor). Cells were treated with 1 mg/mL LPS in the presence or absence of 50 μM BGP-15 for 30 minutes. LPS-induced ROS production was determined by the oxidation of DHR123 (1 μM) to R123, measured with fluorescent microscopy. Cell nuclei were labelled using Hoechst 33342. Representative merged images of three independent experiments are presented. (E) Quantitative analysis of LPS-induced (1 μg/mL) ROS production and the protective effect of BGP-15 (50 μM). Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to control cells; ^###P < 0.001 compared to LPS-treated cells. (F) Effect of BGP-15 on oxidative stress-induced superoxide production in U-251 MG cells in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 compared to control cells, ^##P < 0.01 compared to LPS-treated cells.</p

BGP-15 attenuates oxidative stress-induced DHR123 oxidation and superoxide formation in complexes I-III in WRL-68 and H9c2 cells.

<p>(A) Effect of BGP-15 on oxidative stress-induced DHR123 oxidation in WRL-68 cells. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to control cells; ^#P < 0.05 and ^##P < 0.01 compared to H₂O₂-treated cells. (B) Effect of BGP-15 on oxidative stress-induced DHR123 oxidation in H9c2 cardiomyocytes. Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to control cells, ^#P < 0.05 compared to H₂O₂-treated cells. (C) BGP-15 in chemical reactions does not inhibit DHR123 oxidation induced by H₂O₂ (500 μM). Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to control group. (D) BGP-15 in chemical reactions does not inhibit DHR123 oxidation induced by H₂O₂ (50 μM) and Fe(II)-EDTA (66 μM) (Fenton reaction system). Results are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to the control group. (E) Effect of BGP-15 on oxidative stress-induced superoxide production in WRL-68 cells in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 compared to control cells, ^#P < 0.05 compared to H₂O₂-treated cells. (F) Effect of BGP-15 on the oxidative stress-induced superoxide production in H9c2 cardiomyocytes in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to control cells, ^#P < 0.05 compared to H₂O₂-treated cells. (G) Effect of BGP-15 on mitochondrial DHR123 oxidation using glutamate-malate as substrate and with complex III inhibited by antimycin A. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 compared to the control group. (H) Effect of BGP-15 on mitochondrial DHR123 oxidation using succinate as substrate and with complex IV inhibited by CN^-. Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to the control group.</p

BGP-15 is enriched in mitochondria and reduces membrane potential (ΔΨ) in isolated mitochondria.

<p>(A) Membrane potential enhanced the mitochondrial uptake of BGP-15 (50 μM) in isolated rat liver mitochondria. Uncoupling was found to occur with 50 μM 2,4-dinitrophenol. Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to coupled mitochondria, ^###P < 0.001 compared to the glucose-6-phosphate signal. (B) Mitochondrial membrane potential was monitored by measuring the fluorescence intensity of R123, a cationic fluorescent dye. Isolated rat liver mitochondria, represented by the first arrow, took up the dye in a voltage-dependent manner, resulting in fluorescent quenching. At the second arrow either 1 mM, 2.5 mM or 5 mM BGP-15 was added. A representative plot of three independent concurrent experiments is presented.</p

BGP-15 protects against reactive oxygen species-induced depolarization of mitochondria in WRL-68 cells, as determined by JC-1 and TMRM.

<p>(A) Effect of BGP-15 on H₂O₂-induced mitochondrial membrane depolarization in WRL-68 cells. Cells were exposed to 50 μM H₂O₂ in the absence or presence of 50 μM BGP-15 for 3 hours, then stained with 100 ng/mL of JC-1, a membrane potential-sensitive fluorescent dye. The dye was loaded, and after a 15 minute incubation fluorescent microscopic images were taken using both the red and green channels. The inserts show the homogenous red fluorescence in H₂O₂-treated cells, and the dotted labelling represents the H₂O₂ + BGP-15 treated cells, showing that BGP-15 protected the mitochondrial integrity in the presence of H₂O₂. Inserts are expanded from the area indicated by dashed rectangles. Representative merged images of three independent experiments are presented. (B) Quantitative analysis of mitochondrial depolarization induced by H₂O₂ (50 μM) and its reduction by BGP-15 (50 μM) in WRL-68 cells. Results are presented as the mean ± SEM. ***P < 0.001 compared to control cells, ^#P < 0.05 compared to H₂O₂-treated cells. (C) Effect of BGP-15 on H₂O₂-induced mitochondrial membrane depolarization in WRL-68 cells. Cells were treated with 50 μM H₂O₂ in the absence or presence of 50 μM BGP-15 for 3 hours, then stained with 50 ng/mL of TMRM, a cationic, cell-permeant, red fluorescent dye. After a 15 minutes incubation fluorescent signal was measured by the GloMax Multi Detection System, then remeasured after the application of 1 μM FCCP ΔΨ was calculated as the difference of fluoresescence signal before and after FCCP-treatment. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001 compared to control cells; ^##P < 0.01 compared to H₂O₂-treated cells.</p

Effect of DEA on Akt, GSK-3β and ERK pathways in T24 cells.

<p>T24 cells were treated with increasing concentrations of DEA for 6 hours as indicated. Total cell extracts were analyzed by immunoblotting utilizing anti-total ERK1/2 (ERK), anti-p-ERK1/2 (pERK), anti-Akt (t-Akt), anti-phospho-Akt (p-Akt), anti-GSK (GSK) and anti-phospho-GSK (p-GSK) primary antibodies. The results are presented as representative immunoblots (A) and densitometric analysis of immunoblots in bar diagrams (B). The results are mean ± SD of three independent experiments: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the corresponding control group.</p

The effect of DEA on the expression of BMI1 and on the cell cycle in T24 cells.

<p>(A) T24 cells were exposed to increasing concentrations of DEA for 24-hour intervals. Equal amounts of lysate protein were subjected to gel electrophoresis. Expression levels of BMI1 were monitored by immunoblot assay. GAPDH was used as loading control. The results are presented as representative immunoblots and their densitometric analysis in bar diagram. Data represent mean ± SD of three independent experiments: **p < 0.01 and ***p < 0.001 compared to the corresponding control group. (B) T24 cells were treated with 10 μM of DEA for 24 hours. Cells were harvested, fixed with ethanol and stained with propidium iodide. DNA content was determined using the Muse^™ Cell Analyzer. Graphs demonstrate the percentage of G0/G1phase (dark gray bars), S phase (striped bars) and G2/M phase (white bars). Untreated cells served as controls. Each column represents the average obtained from three independent experiments. Data are presented as the mean ± SD, ***p < 0.001 compared to control.</p

Effect of DEA on cell viability and on colony formation of T24 cells.

<p>(A) T24 cells were exposed to increasing concentrations of DEA for 24 (dark grey bars) and 48 hours (light gray bars). Untreated cells served as controls. Data represent means ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the corresponding control group. (B) For the colony formation assay T24 cells were exposed to increasing concentrations of DEA for 7 days. The results are presented as representative images of the colony formation assay. The colony-forming abilities are also presented in bar diagrams (C). Untreated cells served as controls. The results are mean ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control group.</p

Effect of DEA on activation of apoptosis in T24 cells.

<p>T24 cells were treated with increasing concentrations of DEA for 24 hours to induce apoptosis, then stained with the Muse^™ Annexin V & Dead Cell Reagent, and acquired on the Muse^™ Cell Analyzer. (A) Graphs demonstrate the percentage of living (dark gray bars), early apoptotic (striped bars), late apoptotic (white bars) and total apoptotic cells (light gray bars). Untreated cells served as controls. The results are mean ± SD of three independent experiments performed in at least quadruplicate: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the corresponding control group. In order to investigate the fragmentation of nuclei in T24 cells we treated them with increasing concentrations of DEA for 24 hours. Apoptosis was assessed by Hoechst 33342 staining. (B) Quantification of apoptotic cells was performed by taking the images in random fields and counting cells with strong fluorescence, and condensed or fragmented nuclei. (C) The rate of nuclear fragmentation is also presented in bar diagrams. Each column represents the average obtained from three independent experiments. Data are presented as the mean ± SD, **p < 0.01, ***p < 0.001 compared to control.</p