Trastuzumab and Doxorubicin Sequential Administration Increases Oxidative Stress and Phosphorylation of Connexin 43 on Ser368

Figures

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  Figure 1. Effect of Doxo and Tzb and their association with cytosolic and mitochondrial ROS production. Doxo (1 µM) was administered for 4 h, and Tzb (200 nM) was administered for 20 h. ROS formation was evaluated by means of 2′,7′dichlorofluorescein diacetate (H2DCF-DA) probe in H9c2 cells. ROS production is expressed as mean ± SEM of percentage of DCF positive cells of at least three independent experiments, each performed in duplicate (A). Mitochondrial superoxide production was measured by means of the probe MitoSOX Red in H9c2 cells through flow cytometry analysis, and it is expressed as mean ± S.E.M. of MitoSOX positive cells percentage of at least three independent experiments each performed in duplicate (C). Data were analysed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. * p < 0.05 and ** p < 0.005 vs. non-treated cells. Panels (B,D) report representative histograms for the flow cytometry analysis of DCF and MitoSOX, respectively.

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  Figure 2. Doxo and Tzb and their association effect on mitochondrial membrane depolarization. Doxo (1 µM) was administered for 4 h, and Tzb (200 nM) was administered for 20 h. Mitochondrial membrane potential was evaluated by flow cytometry analysis with Tetramethylrhodamine ethyl ester (TMRE), a cell permeant, positively charged, red-orange dye, which comes in and accumulates in the mitochondria in inverse proportion to the membrane potential. The low value of TMRE+ cells percentage means indicates that the TMRE dye was not trapped in the mitochondrial membrane due to its depolarization. Results are expressed as mean ± S.E.M. of fluorescence intensity of at least three independent experiments, each performed in duplicate (A). Data were analysed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005, *** p < 0.001 and **** p < 0.0001 vs. non-treated cells. Panels (B) reports representative histograms for the flow cytometry analysis.

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  Figure 3. Effect of Doxo and Tzb and their association with calcium homeostasis. Doxo (1 µM) was administered for 4 h, and Tzb (200 nM) was administered for 20 h. Mitochondrial calcium pool was evaluated on H9c2 cells in calcium-free medium in the presence of FCCP (50 nM) (panel (A)). Panel (B) shows the intracellular calcium content in calcium-free medium evaluated by means of Ionomycin (1 μM). Results are expressed as mean ± S.E.M. of percentage of delta (% Δ) increase in FURA 2 ratio fluorescence (340/380 nm) from at least three independent experiments, each performed in duplicate. Data were analysed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005 vs. non-treated cells, # p < 0.05 vs. Doxo-treated cells.

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  Figure 4. Doxo and Tzb and their association effect on apoptosis. Doxo (1 µM) was administered for 4 h, and Tzb (200 nM) was administered for 20 h. H9c2 cells were stained by PI, and individual nuclei fluorescence was measured by flow cytometry. Results are expressed as mean ± S.E.M. of percentage of hyplodiploid nuclei from at least three independent experiments, each performed in duplicate (panel (A)). Panels (B) reports the representative histograms for the flow cytometry analysis. Cytosolic cytochrome c level was detected by flow cytometry analysis. Results are expressed as mean ± S.E.M. of percentage of cytochrome c positive cells from at least three independent experiments each performed in duplicate (panel (C)). Data were analysed by variance test analyses, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005 and *** p < 0.001 vs. non-treated cells, ## p < 0.005 vs. Doxo-treated cells. Panel (D) reports the representative images of flow cytometry analysis of cytochrome c expression.

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  Figure 5. Doxo and Tzb and their association effect on Cx43 and pCx43 expression. Doxo (1 µM) was administered for 4 h, and Tzb (200 nM) was administered for 20 h. Cx43 and pCx43 expression was checked through flow cytometry analysis. Results are expressed as mean ± S.E.M. of percentage of Cx43 (A) and pCx43 (C) positive cells from at least three independent experiments, each performed in duplicate. Data were analysed by variance test analysis, and multiple comparisons were made by Bonferroni’s test. ** p < 0.005 vs. non-treated cells, ° p < 0.005 vs. Tzb-treated cells. Panels (B,D) report representative images of flow cytometry analysis of Cx43 and pCx43 expression, respectively.