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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

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

Fig. 4

P2XR4 activity protects from ROS. A Left: Representative confocal image showing ROS production (DCFDA fluorescence) in a single cell assay. TOP A-498 cell control treated with vehicle (DMSO) or exposed for 15 min to 5-BDBD (bottom). Right: cells stained with DCFHDA green were boxed and fluorescence quantified were Graphical represented as relative fluorescence intensity of ROS in the square area. B Graphical representation of mean DCFDA fluorescence intensity (recorded as λEx/Em = 495/529 nm) reported as fold change over control vehicle in SNC-12 and A-498 cells, treated for 10 min with 0.5 μM 5-BDBD or positive control (500 μM H2O2) or negative controls (10 μM CCCP, protonophore m-chlorophenylhydrazone). C C11-BODIPY581/591 was used to index lipid peroxidation. SNC-12 or A-498 cells were incubated with 2.5 µM C11-BODIPY.581/591 for 15 min after exposure to 5-BDBD (0.5 μM), H2O2 (500 μM), and negative control 10 μM CCCP (protonophore m-chlorophenylhydrazone) for 10 min. Data are reported as fold change to vehicle treated cells. D, E GSSG and GSH colorimetric assay in A-498 and SNC-12 cells treated for 15 min with vehicle or with 5-BDBD. Data are reported as millimol of GSH or GSSG / µg of protein extracts. F Western blot of protein extracts from A-498 and SNC-12 cells collected at different time points from 5-BDBD treatment stained with Catalase antibody. Tubulin was used as loading control. G Representative confocal images showing ROS production (DCFDA fluorescence) in scramble transfected control A-498 cells (Scr) or in silenced P2X4 clone (siP4#1). The mean relative fluorescence intensity measured (MFI) in the squared area is indicated. H qRT-PCR dosage of P2X4 mRNA in scramble control A-498 cells (Scr) and in two different silenced clones, siP4#1 and siP4#2. I Western Blot analysis of proteins from scramble control A-498 cells (Scr) and in two silenced clones siP4#1 and siP4#2 with P2XR4 antibodies and GAPDH as loading control. J Cell proliferation assay in A-498 scramble transfected cells (Scr), and in siPX4 clones 1 and 2 assessed by MTT at different days of cultures (n = 3) reported as OD at 570 nm. K MitoTracker Red staining for mitochondria in scramble transfected A-498 cells (Scr) and siP4#1 clone (scale bar = 10 μm). L Relative fluorescence intensity for MitoTracker Red along the diameter of a siP4#1 single cell. M Oxygen consumption rate (OCR) measurements in scramble transfected A-498, (Scr) and in siP4#1 clone with the Mito Stress Test. Data B, C, D, E, H and J M represent as mean ± SD of three independent experiments performed in triplicate. Student’s t-test ** p < 0.01.***p < 0.001

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