Fig 2: Purinergic pharmacology and expression of the receptor involved in the electrical response in SKOV-3 cells. (A) A battery of different agonists (all applied at 3 µM) for purinergic receptors was tested in cells held at -40 mV, and the peak current amplitude for each agonist was normalized against that obtained by applying ATP. This resulted in the potency sequence illustrated in graph (B) where data obtained in 50 cells are summarized. (C) The upper panel shows the PCR analysis for P2Y receptors sensitive to ATP/UTP; P2Y2, P2Y4 and P2Y6 receptor subtypes. In SKOV-3 cells, bands with the expected size were obtained for P2Y2 and P2Y4 subtypes but not for P2Y6 (mRNA from HELA cells was used as positive control, and -RT indicates the corresponding reaction control without reverse transcriptase). The images show immunorecognition for P2Y2 or P2Y4 receptor proteins in SKOV-3 cells observed by epifluorescence (P2Y receptor in green and nuclei in red labeled with propidium iodide). Scale bar 10 µm. (D) Traces illustrate the effect of AR-C118925, NF340, or MRS2179, antagonists of P2Y2, P2Y1 or P2Y11 receptor, respectively. Each set of records corresponds to the current response elicited by ATP (3 µM; black trace) alone or in the presence of an antagonist (gray trace) as indicated. (E) The graph summarizes the results obtained in experiments as in (D). Each bar corresponds to the current amplitude in every condition (n = 16) normalized against the current obtained by applying ATP alone. The graph also shows results of using UTP as agonist and AR-C118925 as antagonist (n = 16; *p < 0.05).

Fig 3: Co-localization of P2Y2 receptor and KCa3.1 channel in human ovarian carcinoma. Expression of P2Y2 receptors and KCa3.1 channels in slices from human ovarian carcinoma evaluated immunohistochemically. Biopsies from ovarian carcinoma tissue were collected, processed, and stained with hematoxylin-eosin and/or labeled with KCa3.1- and P2Y2-specific antibodies that were revealed by second antibodies coupled to green and red fluorescent dyes, respectively. Nuclei in blue labeled with DAPI. Positive co-expression was detected in ovarian neoplastic cells (see merged image), whereas no signal was observed in control assays in which the primary antibody was omitted. Expression of KCa3.1 was also observed in stromal cells that did not show neoplastic morphology (*); however, P2Y2 did not co-localize with KCa3.1 in these cells. Patient 1, IC16-532-6.

Fig 4: Comparison of P2Y2 protein clustering in AsPC-1 cells under control, agonistic and antagonistic conditions. (a) Density of P2Y2 proteins under control, agonistic and antagonistic conditions. (b) Percentage of P2Y2 detected as monomers under control, agonistic and antagonistic conditions. (c) Percentage of P2Y2 forming dimers under control, agonistic and antagonistic conditions. (d) Percentage of P2Y2 receptors forming clusters of more than 5 proteins under control, agonistic and antagonistic conditions. (e) Number of P2Y2 proteins per cluster under control, agonistic, and antagonistic conditions. Control conditions depicted in grey with circular points, agonist in yellow with triangular points and antagonist in blue with square points. Significance values from paired Wilcoxon sum tests adjusted p values >0.05 = ns, <0.05 = *, <0.01 = **, <0.001 = ***. n = 25, 27, and 26 ROIs, respectively.

Fig 5: P2Y2 and KCa3.1 protein expression and electric response to agonists in esiRNA transfected SKOV-3 cells. (A) Analysis by immunocytochemistry after 48 h of esiRNA treatment in control (CNT) and P2Y2-esiRNA-treated groups. Panels show the fluorescence signal for DAPI (blue) in the first column, the signal obtained with a specific antibody against P2Y2 receptor protein (in green) in the second column, and the corresponding merged image. (B) Similar analysis was made in KCa3.1-esiRNA-treated cells using an antibody against KCa3.1 channel protein (in A and B bar = 50 µm). (C) Traces illustrate current response elicited by agonists (all 3 µM) or 1-EBIO (300 µM) in SKOV-3 cells 24 h after esiRNA transfection. In (D and E) traces illustrate current responses in SKOV-3 cells 24 h and 72 h after esiRNA transfection, respectively. Currents were normalized against the ATP-elicited response in the CNT group (18–19 cells in each condition). Every set of bars corresponds to either CNT, P2Y2-esiRNA, or KCa3.1-esiRNA as indicated in the group for ATP application (*p < 0.05, experimental condition vs. respective CNT).