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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: Therapeutic targeting of P2X4 receptor and mitochondrial metabolism in clear cell renal carcinoma models

Fig. 5

P2XR4 protects mitochondrial membrane from oxidation and calcium overload. A Determination of mitochondrial membrane potential (ΔΨm) in A-498 (top) and SNC-12 cells (bottom) with JC-1 staining, using fluorescence cell sorting. The cells were treated for 15 min with CCCP (positive control), 5-BDBD, or vehicle. The high mitochondrial membrane potential (ΔΨm), in red corresponds to dimers of JC-1, and the low ΔΨm in green corresponds to the JC-1 monomer. Percentages of red and green potentials (ΔΨm) are indicated in each quadrant. B Representative images of A-498 and SNC-12 cells incubated with or without 5-BDBD for 15 min and stained with JC-1. Red staining indicates high mitochondrial membrane potential and green staining indicates low potential (ΔΨm). Scale bar = 10 µm. C Representative image of a single cell assay of A-498 cells treated with 5-BDBD for 15 min (upper panel) or vehicle- (lower panel) stained with MitoTracker green and Rhodamine-AM red as indicators of mitochondrial calcium accumulation. Correlation between green and red fluorescence intensity in single cell assay with Pearson test; R = 0.64 (scale bar 10 µm). D Representative confocal image of Rhodamine-AM (red) stained mitochondrial calcium in A-498 cells treated for 15 min with vehicle (control) or 5-BDBD (scale bar 10 µm). E Flow cytometry quantification of mitochondrial calcium accumulation by Rhodamine-2AM (red) in vehicle control, A-498, and SNC-12 cells, or cells exposed to 2 mM Ca2+ with or without 5-BDBD. The percentage of Rhodamine-2 AM positive mitochondria (red) is indicated in the corresponding quadrant. F Western blot analysis of pro-Caspase 9 and 3 in protein extracts from A-498, SNC-12, and control HRE cells treated with 5-BDBD for different times. G Early and late apoptosis in A-498 and SNC-12 cells treated with 5-BDBD or vehicle for 24 h stained with FICT-Annexin V and PI and analyzed by flow cytometry. H Quantification of early and late apoptotic cells. Data are mean ± SD. of three independent experiments performed in triplicates ***p < 0.001. Statistical analysis was performed using one-way ANOVA followed by Turkey’s post-hoc test. I Dose response curve and IC50 for 5-BDBD determined by MTT assay reported as vitality / control, A-498, SNC-12, HRE; and 786–0 cells. Data are presented as the mean ± SD of three independent experiments performed in triplicates

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