Fig. 3
Dopamine induced p-Ser637DRP1 depletion, mitochondrial fragmentation and apoptotic Cytochrome c re-localization is attenuated by Calcineurin inhibition. A SH-SY5Y cells are treated with DA (200 µM) or as mentioned for 16 h and total cell lysate is subjected to SDS PAGE followed by immunoblotting for the mentioned proteins. Bar graphs represent mean intensity value of p-Ser637DRP1 normalized by total DRP1. Immunoblots are representative of at least 3 different experiments. **P ≤ 0.05 as compared to control, one way ANOVA followed by Tukey’s multiple comparison test. B After the mentioned treatment for 16 h, Calcineurin (CaN) activity from the SH-SY5Y cell lysate is measured and the mean activity is represented. *P ≤ 0.05. C SH-SY5Y cells are treated for 16 h and cytosolic free Ca2+ is measured by Fluo-4-AM intensity. Mean intensity of each group is represented as bar graph. *P ≤ 0.05 as compared to control, #P ≤ 0.05 when compared to DA treated group. D After treatment, as mentioned, mitochondrial morphology of SH-SY5Y cells is analyzed from the captured images. Images are representative of 3 different experiments and at least 30 cells were analyzed. Scale bar 10 µm. Mitochondrial morphology is classified as mentioned earlier and mean proportion of filamentous, rod shaped or punctate mitochondria is represented in the bar graphs. *P ≤ 0.05, **P ≤ 0.01 as compared to control. E Control or MEF cells expressing DRP1K38A/dominant negative Calcineurin (ΔCnAH151Q) are treated as mentioned, for 16 h and mitochondria are imaged. Mitochondrial classification and quantification are done as mentioned above. *P ≤ 0.05 as compared to control. Scale bar 10 µm. F SH-SY5Y cells are treated with DA or DA + FK-506 for 16 h. Fixed cells are stained for ATP5a and Cytochrome c. Co-localization is quantified by measuring Mander’s coefficient and mean values are represented as bar graph. At least 30 cells were analyzed from 3 independent experiments. *P ≤ 0.05, **P ≤ 0.01 as compared to control, #P ≤ 0.05 as compared to DA treated group. G SH-SY5Y cell death is analyzed by propidium iodide (PI) or trypan blue staining, after DA or DA + FK-506 treatment for 24 h. *P ≤ 0.05, ***P ≤ 0.001when compared to control group; #P ≤ 0.05 and ##P ≤ 0.01 when compared to DA treated group. Bar graphs represent mean ± SEM. N = at least 3 for each group. P values are calculated by one way ANOVA followed by Tukey’s multiple comparison test unless mentioned otherwise