Fig 1: Crude LPS and purified SAA induced Panx1 release in macrophage and monocyte cultures. (a,b) Extracellular release of Panx1 by murine macrophages. Murine macrophage-like RAW264.7 cells were stimulated with LPS or SAA at indicated concentrations for 16 h. The macrophage-conditioned culture medium was subjected to differential centrifugations, and each fraction was assayed for Panx1 by Western blotting analysis. Note Panx1 was found in the LPS- or SAA-stimulated macrophage-conditioned medium (“Cell Medium”), and the supernatant of the 20,000 × g centrifugation. (c,d) Extracellular release of Panx1 by primary human monocytes. Human PBMCs were stimulated with crude LPS at indicated concentrations for different time periods, and extracellular Panx1 levels were determined by Western blotting analysis. The relative optical intensity of the 48-kDa (Panx1) and 12-kDa (Panx1Δ) band was measured, and expressed as an arbitrary unit (AU).
Fig 2: An exogenous bacterial endotoxin (LPS) and an endogenous inflammatory cytokine (SAA) up-regulated Panx1 expression in macrophage (Mϕ) and monocyte (MC) cultures. Murine macrophage-like RAW264.7 cells (Panel a), differentiated human macrophages (Panel b), human peripheral blood mononuclear cells (MC, Panel c), or primary murine peritoneal macrophages (Panel d) were stimulated with crude LPS or SAA at indicated concentrations for indicated time periods, and the cellular Panx1 levels were measured by Western blotting (Panel a,b,c) or immunocytochemistry (Panel d), respectively. A house-keeping gene product β-actin was used as a loading control to estimate the relative Panx1/β-actin (P/A) ratio in macrophage or monocyte cultures.
Fig 3: Panx1 mimetic peptide (10Panx) exacerbated lethal sepsis. Male Balb/c mice were subjected to CLP-induced sepsis, and intraperitoneally administered with control saline (0.2 ml/mouse) or 10Panx peptide at indicated doses at +2, +24 h post CLP. Animal survival was assessed for up to two weeks, and the Kaplan-Meier method was used to compare the differences in mortality rates between groups. *P < 0.05 versus “+Saline” group.
Fig 4: Altered expression of Panx1 during lethal sepsis. Balb/c mice were subjected to lethal sepsis by a surgical procedure termed cecal ligation and puncture (CLP), blood and various tissues were harvested at 24 h post CLP to measure Panx1 protein (Panel a,b) or mRNA levels (Panel c) by Western blotting and real-time RT-PCR, respectively. Serum Panx1 levels were measured by the optical intensity of the 48-kDa band on the Western blot, and expressed as the % of the optical band intensity of corresponding total proteins on SDS-PAGE gels. *P < 0.05 versus normal “-CLP” control.
Fig 5: Panx1 mimetic peptide 10Panx enhanced LPS-induced macrophage hemichannel activation. (a,b) 10Panx elevated the LPS-induced Lucifer Yellow dye uptake. RAW264.7 cells were stimulated with LPS (1.0 µg/ml) in the absence or presence of 10Panx at indicated concentrations for 16 h, and subsequently incubated with Lucifer Yellow (LY, 1.0 mg/ml) for 15 min. Following fixation and three extensive washes, the number of cells with diffused fluorescent signals was counted under a fluorescence microscope, and expressed as a percentage of total cell numbers (DAPI-stained nuclei) in six fields. (c) 10Panx enhanced the LPS-induced ATP release. RAW264.7 cells were cultured in serum-free DMEM medium, and stimulated with LPS (1.0 µg/ml) in the absence or presence of Panx1 mimetic peptide at indicated concentrations, and the cell-conditioned culture medium was collected and subjected to ATP measurement. *P < 0.05 versus “-LPS” Control; #P < 0.05 versus “+LPS” control.
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