Fig. 3

P2XR4 receptor regulates intracellular calcium. Cytosol Ca²⁺ dosage in live A-498 cells traced by Fura-2 AM over the time. A Baseline reading in Ca²⁺ free conditions were established for 2 min, then cells were recorded for additional 2 min without or with P2XR4 inhibitor (5-BDBD at different concentration green lines). B Intracellular calcium response to 5-BDBD, or inositol 3 phosphate receptor inhibitor (IP3Ri), or lysomotropic inhibitor (L-L-MA) of A-498 cells incubated in Ca²⁺-free medium. C Intracellular calcium levels in cells stimulated with 2 mM Ca²⁺-containing medium in presence of either a P2XR7 inhibitor (A804598) or 5-BDBD or combination of both. D Intracellular calcium levels in cells stimulated with 3 mM ATP and 2 mM Ca²⁺-containing medium in presence of either a P2XR7 inhibitor (A804598) or 5-BDBD or both. E Box plot of intracellular calcium in A-498 cells following inhibition of mitochondria (CBX), P2XR7 (A804598), P2XR4 (5-BDBD), or IP3Ri, as indicated. Data are reported as ratio between F and F0 (340/380 nm fluorescence of Fura 2-AM) recorded in controls, n = 51 cells, A804598, n = 50 cells, 5-BDBD n = 39 cells and IP3Ri inhibitor n = 45 during the 2 min of stimulation. P-values were calculated by Mann–Whitney U **p < 0.01. F Pharmacological blockage of P2XR4 results in mitochondrial morphology change. Representative confocal image of SNC-12 and A498 cells mitochondria stained with MitoSox (red) and DAPI for nuclei (blue). After treatment with 5-BDBD for 10 min the mitochondria appeared fused in filaments in A-498 cells or with large cristae in SNC-12 cells. Scale bars = 10 µm. G Representative flow cytometry analysis of mitochondria stained with MitoSox (red) from A-498 cells after different time of 5 µM 5-BDBD treatment, as indicated. The percentage of positive cells is reported in each quadrant. H Oxygen consumption rate (OCR) with the Mito Stress Test kit in HRE cells incubated with or without 5 µM 5-BDBD over the time. I and J OCR measurements in A-498 and SNC-12 cells with or without 5 µM 5-BDBD. K OCR in 786–0 cells with or without 5 µM 5-BDBD. L and M OCR vs extracellular acidification rate (ECAR) in A498 and SNC-12 cells treated with 5-BDBD at different concentrations, as indicated. Data represent mean ± SD of three independent experiments performed in triplicate