Psb0739

Paclitaxel, ADP, Apyrase, Tween-80 and polyethylene glycol-300 (PEG-300) were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Cisplatin and erlotinib were obtained from Selleckchem (Pittsburgh, PA, USA). Gemcitabine was obtained from Eli Lilly (Indianapolis, IN, USA). Ticagrelor was obtained from Sigma-Aldrich, Selleckchem and Pure Chemistry Scientific Inc. (Burlington, MA, USA). PSB-0739 and MRS 2179 were from Tocris Bioscience (Bristol, UK). Cultrex basement membrane Type-3 was from Trevigen, Inc. (Minneapolis, MN, USA). Matrigel was obtained from Corning Life Sciences (Corning, NY, USA)

CX3CR1^+/GFP mice were subjected to two-photon excitation laser scanning microscopy under ketamine and xylazine (100 mg kg⁻¹ and 10 mg kg⁻¹, respectively) anesthesia. A cranial window (1.8 mm in diameter) was made in the somatosensory cortex, and the dura was carefully removed. P. gingivalis, S. mitis and E. coli were prepared at 3.6 × 10⁵ CFU ml⁻¹ in artificial cerebrospinal fluid (ACSF) with 0.5% rhodamine-dextran (Invitrogen, Carlsbad, CA, USA). ATP (10 mM; Sigma-Aldrich Japan K.K., Tokyo, Japan), UDP (10 mM; Sigma-Aldrich), the bacteria mentioned above, P. gingivalis LPS (100 μg ml⁻¹; Invivogen), and fMLP (10 μM, Sigma Aldrich) were injected into the brain (50–100 μm in depth) through a glass capillary (tip diameter 3–6 μm). PSB0739 (a selective P2Y₁₂R inhibitor, 1 μM; Tocris Bioscience, Bristol, UK), MRS2578 (a selective P2Y₆R inhibitor, 1 μM; Sigma-Aldrich) and apyrase (5 U ml⁻¹; Sigma-Aldrich) diluted in ACSF were applied to the surface of the somatosensory cortex for 30 min, and then images were obtained with 16 × 0.8 NA water-immersion lens and a Spectra Physics Mai-Tai IR laser tuned at 920 nm for two-photon excitation of GFP using multiphoton confocal microscope (A1RMP, Nikon, Tokyo, Japan). Z-stack images were taken 1 μm apart from 15 μm above to 15 μm underneath the tip of the capillary every 5 min for 40 min.

ADP, ATP, UDP, and UTP were purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-human Bcl-2, total ERK, phospho-ERK antibodies, Rabbit anti-human nucleolin antibody, and ERK inhibitor U0126 were purchased from Cell Signaling Technology (Beverly, MA). P2Y1, 12, 13 agonist 2-MeSADP, P2Y1 selective inhibitor MRS2179, P2Y12 potential inhibitor PSB0739, P2Y13 competitive inhibitor MRS2211 were purchased from Tocris (Bristol, UK). Mammalian expression plasmid pReceive-M29 coding for eGFP-nucleolin fusion protein was purchased from GeneCopoeia (Germantown, MD).