Protective effect of Mel on Oxa induced intracellular accumulation of superoxide anion and oxidative stress.

<p>Superoxide anion production was monitored by DHE florescent dye. Data were represented as mean ± SE (n = 6) and values were expressed as DHE stained positive cells. Significant difference (***P ˂ 0.001) was indicated as compared to control and significant difference (###P ˂ 0.001) was shown as compared to Oxa treated groups. The DCF fluorescence integrated density was analyzed with Image J software (1.50 version, NIH, USA). Five to six random images were captured at 20x magnification. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. Superoxide ion accumulation was quantified with at least three independent experiments.</p

Protective effect of Mel on DNA damage induced by Oxa.

<p>Representative images (A) of comet assay on SH-SY5Y cells after exposure to Oxa (50 μM). Oxa incurred DNA damage leading to more number of comet formation compared to Mel pre-treatment cells. DNA damage in cells was quantified as olive tail moment (B) and tail length (C). Significant difference (***P ˂ 0.001) was indicated in comparison to control and significant difference (##P ˂ 0.01) was shown when compared to Oxa treated groups.</p

Representative immunoblots for Bax and Cyt c localization.

<p>Protective effect of Mel on Oxa induced Cyt c release into cytosol and Bax translocation from cytosol to mitochondria were evaluated in SH-SY5Y cell. Cells were pre-treated with 10 μM Mel for 1 h followed by with and without 50 μM Oxa for 24 h. COX IV and β-actin levels were monitored to verify equal amount of protein loading.</p

Effect of Mel pre-treatment on Oxa induced mitochondrial membrane depolarization in SH-SY5Y cells.

<p>Membrane depolarization was monitored by TMRE florescence integrated density. Data were represented as mean ± SE (n = 6) Significant difference (***P ˂ 0.001) was indicated as compared to control and significant difference (###P ˂ 0.001) was shown as compared to Oxa treated groups. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. The TMRE fluorescence integrated density was analyzed with Image J software (1.50 version, NIH, USA). Membrane potential was measured with at least three independent experiments.</p

Protective effect of Mel on Oxa induced pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) signal in SH-SY5Y cells.

<p>The quantification of immunoreactive bands for Bax and Bcl-2 realative to GAPDH and Bcl-2/Bax ratio were determined. Data were represented as mean ± SE (n = 6) and relative expression levels were quantified from four independent experiments. Significant difference (***P ˂ 0.001) was indicated as compared to control and significant difference (###P ˂ 0.001) was shown as compared to oxa treated groups. GAPDH level were monitored to verify equal amount of protein loading.</p

Protective effect of Mel on Oxa induced cytotoxicity assessed by mitochondrial reduction of MTT to formazan.

<p>Percentage of live cells were calculated and plotted as histogram. Data were represented as mean ± SE (n = 6) Significant difference (*P ˂ 0.05 and ***P ˂ 0.001) was indicated as compared to control and significant difference (#P ˂ 0.05 and ###P ˂ 0.001) was shown with compared to different concentration of Oxa treated groups. Cell viability was measured from at least three independent experiments.</p

Inhibitory effect of Mel on Oxa induced intracellular accumulation of ROS and oxidative stress.

<p>ROS level were monitored by DCFHDA florescent dye. Data were represented as mean ± SE (n = 6) Significant difference (***P ˂ 0.001) was indicated as compared to control and significant difference (###P ˂ 0.001) was shown as compared to Oxa treated groups. ROS measurement was monitored by DCF florescence integrated density. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. The DCF florescence integrated density was analyzed with Image J software (1.50 version, NIH, USA). ROS accumulation was measured with at least three independent experiments.</p

Protective effect of Mel on Oxa induced PARP inactivation in SH-SY5Y cell.

<p>Data were represented as mean ± SE and relative expression levels were quantified from four independent experiments. Significant difference (**P ˂ 0.01) was indicated when compared to control and significant difference (#P ˂ 0.05) was shown in comparison to the Oxa treated groups. The quantification of immunoreactive bands for PARP and cleaved PARP relative to GAPDH and cleaved PARP/total PARP ratio were determined. GAPDH level were monitored to verify equal amount of protein loading.</p

Protective effect of Mel on Oxa induced cytotoxicity in SH-SY5Y cells.

<p>Cytotoxicity was determined by LDH activity. Values were expressed as μU/ml of LDH activity. Data were represented as mean ± SE (n = 6) Significant difference (***P ˂ 0.001) was indicated as compared to control and significant difference (###P ˂ 0.001) was shown as compared to different concentration of Oxa treated groups. Cytotoxicity via LDH activity was measured with at least three independent experiments.</p

Effect of Mel pre-treatment on Oxa induced cytotoxicity by live cell assay in SH-SY5Y cells.

<p>Live cell assay was done by utilizing calcein fluorescent probe. Calcein AM can enter into cells and stains only live cells. Oxa treated cells showed shrinkage, round up and clumping indicative of loss of cell viability and progression towards death. Live Cell Assay was performed with at least three independent experiments.</p