Fig 2: The expression of P2X4R in microglia increased after ICH. A Protein expression of P2X4R was analyzed by western blot in the hematoma hemisphere at different time points after ICH (n = 5). B Quantification of P2X4R at the protein level. C Immunofluorescence staining of P2X4R/GFAP, P2X4R/NeuN-1, and P2X4R/ Iba-1 in the murine brain 7 days after ICH (n = 3). Data are expressed as the means ± SEM. *P < 0.05. **P < 0.01. ***P < 0.001. Scale bar = 50 µm

Fig 3: P2X4 immunoexpression in P0 rats subjected to hypoxia (Hy) at different time points. P0 rats subjected to Hy and sacrificed at 4 (Hy4 h), 24 (Hy24 h), 72 (Hy72 h), 7D (Hy7 d) and 14D (Hy14 d). Note P2X4 immunofluorescence in amoeboid microglial cells overlapping with OX42 fluorescence is noticeably enhanced in the hypoxic rats Hy4 h (A/Ba-c), Hy24 h (C/Da-c), Hy72 h (E/Fa-c), and Hy7 d (H/Ia-c) when compared with the matching controls (Con4 h, Con24 h, Con72 h, Con7 d and Con14d). Scale bar = 20 μm.

Fig 4: Colocalization of P2X4 receptors with endolysosomal markers. rP2X4-EGFP receptors were expressed in NRK cells, and live cells were imaged by confocal fluorescence microscopy 48 h post transfection after co-labeling lysosomes with a variety of different markers. These include (A) LAMP1-mCherry (LAMP1-mCh), which was co-transfected with rP2X4-EGFP, and (B) Texas Red-labeled Dextran (DexTR) incubated for 5 h and chased for 2 h at 37 °C. (C) Calculation of Pearson’s coefficient showed the high degree of colocalization between rP2X4 and LAMP1-mCh/DexTR. Results are the mean ± SEM from three independent experiments, total number of cells, n = 40. (D) Cells were incubated with Lysotracker (50 nM) for 10 min at 37 °C and, in (E,F), with a fluorescent substrate of cathepsin B, Magic RedTM (MR) for 5 min. Cells in (F) were imaged using TIRF microscopy to show only those rP2X4- and MR-positive compartments located within ~100 nm of the plasma membrane. Arrowheads indicate examples of compartments that are labeled only by rP2X4 or MR. Scale bars are 10 µm (5 µm for enlargements).

Fig 5: The BK channel-dependent membrane translocation of the P2X4Rs after morphine stimulation in microglia.(a) A scatter plot of both the ATP (3 mM)-induced currents and BK currents at +30 mV in the lamina I spinal microglia after 5 days of saline or morphine (10 mg kg−1) administration (two-tailed Spearman's test, r=0.8543, P<0.0001, n=13 for Saline, n=13 for Morphine, from four mice each). (b) Typical traces of ATP (3 mM)-induced currents in the MG6 after 24 h of morphine (10 μM) stimulation. Naloxone (Nal, 10 μM) or paxilline (Pax, 2 μM) was added 1 h prior to morphine treatment. (c) The peak amplitude of the ATP-induced inward currents at a holding potential of −60 mV (n=7/8/10/10 cells). (d) An immunoblot to examine the membrane translocation of P2X4Rs following stimulation with morphine (10 μM) for 24 h in primary microglia. The cells were exposed to morphine in the presence or absence of naloxone (Nal, 10 μM) or paxilline (Pax, 2 μM). (e,f) The P2X4R levels in the membrane fraction (e) or total lysates (f) were quantified relative to the control. The data represent the means±s.e.m. ***P=0.0005 (Control versus Morphine), 0.0003 (Morphine versus Morphine+Naloxone), 0.0001 (Morphine versus Morphine+Paxilline), F3, 31=10.87, a one-way ANOVA followed by the Tukey's post hoc test (c). *P=0.0281 (Control versus Morphine), **P=0.0032 (Control versus Morphine), 0.0010 (Morphine versus Morphine+Naloxone), F3, 24=7.824, a one-way ANOVA followed by the Tukey's post hoc test (e). **P=0.0057 (Control versus Morphine), F3, 24=4.956, a one-way ANOVA followed by the Tukey's post hoc test (f). Scale bar, 50 pA and 100 ms (b).