Figure 4
From: P2X4 deficiency reduces atherosclerosis and plaque inflammation in mice
P2X4-deficiency reduces expression release of inflammatory cytokines. Atherosclerotic aortic roots from P2X4−/− LDLR−/− mice (n = 24) and P2X4+/+ LDLR−/− mice (n = 15) after 16 weeks of high-cholesterol diet were stained for FLICA-positive cells (FLICA-fmk, green) and cell nuclei (DAPI, blue). Representative images are shown (A). Frequency of FLICA-positive cells per DAPI-positive cells was analyzed (B). RNA was isolated from atherosclerotic lesions from aortic arches of P2X4−/− LDLR−/− mice (n = 12) and P2X4+/+ LDLR−/− mice (n = 13). Two-step multiplex TaqMan RT-PCR was performed to determine expression of cytokines and adhesion molecules. Expression fold change was calculated by ddCt method, results were referred to β-Actin as the housekeeping gene (C). BMDMs were isolated from the bone marrow of 8-week-old P2X4-competent (n = 5) and P2X4-deficient mice (n = 5). Fully differentiated BMDMs were first stimulated with 100 ng/mL LPS for 4 h, followed by stimulation with either 100 µM or 5 mM ATP for 1 h. Multiplex fluorescence-encoded beads assay was performed to determine concentrations of inflammatory cytokines (D). Results are presented as mean ± SEM. Statistical significance was calculated using Shapiro–Wilk Test followed by an unpaired t-test for parametric or Mann–Whitney-U-test for non-parametric data. *p < 0.05; **p < 0.01; ***p < 0.001.
