Fig 1: Activation of the PKC-Erk1/2-NOS1 axis is related to hyperpermeability in HG-treated hRECs. (a–c) hRECs were treated with high glucose (HG, 25 mM) as the indicated time periods. MN-treated hRECs were used as the treatment control (Supplementary Fig. 5a). Cells were then lysed and total protein was extracted for immunoblotting with anti-PKC (a), anti-phospho-Erk1/2Tyr202/Tyr185 (b) and anti-NOS1 (c) antibodies. Data are presented mean ± SD. n = 3. *p < 0.05 vs 0 h. (d). hRECs were cultured for 24 h under HG conditions in the presence or absence of PKC inhibitor Go 6976. Total cellular protein was extracted, and 50 µg was used for evaluation of PKC kinase activity. Data are presented mean ± SD. n = 3. *p < 0.05 vs CTL; #p < 0.05 vs HG. (e). hRECs were cultured for 24 h in HG with or without Go 6976 (500 nM). Cells were then lysed and total protein was extracted for immunoblotting with anti-phospho-Erk1/2Tyr202/Tyr185 and anti-NOS1 antibodies. The effects of Go 6976 on Erk activation were explored in hRECs under normoglycemia condition (Supplementary Fig. 5b). Data are presented mean ± SD. n = 3. *p < 0.05 vs CTL. (f). Cells were grown on Transwell filters, and treated for 24 h in HG with or without Go 6976 (500 nM). The permeability of monolayer cells was evaluated using FITC conjugated albumin. (g). hRECs were cultured for 24 h in HG with or without Go 6976 (500 nM). Apoptotic cell death was determined using the Cell Death Detection ELISA. f and g. Data are presented mean ± SD. n = 5. *p < 0.01 vs CTL; #p < 0.05 vs HG. Full length blots of a, b, c and e are provided in Supplementary Fig. 6. In b and e, stripped membranes from phospho-Erk were reblotted for total Erk.
Fig 2: VEGF-mediated activation of the p38MAPK/STAT1 signaling via VEGFR2 involves increase of apoptosis in HG-treated hRECs. (a). In cultured hRECs, the siRNA that targets to human VEGFA gene was introduced at 3 different concentrations (siVEGF-1: 5 nM, siVEGF-2: 10 nM, siVEGF-3: 20 nM). Control siRNA (siCTL, 20 nM) that does not target any human gene was used as the transfection control. 24 h after transfection, cells were exposed to high glucose (HG, 25 uM) for 24 h. Cells were then lysed and total cellular protein was extracted for immunoblotting with anti-VEGF, anti-phospho-VEGFR1Tyr1213, and anti-phospho-VEGFR2Tyr1175 antibodies. (b). In VEGF knockdown cell (siVEGF, 20 nM), effect of HG on PKC activity was evaluated using PKC Kinase Activity Assay. (c). In VEGF knockdown cell (siVEGF, 20 nM), immunoblot assay was performed to investigate the effects of HG on the level of phospho-Erk1/2Tyr202/Tyr185 and NOS1 expression. (d). In VEGF knockdown cell (siVEGF, 20 nM), immunoblot assay was performed to show the effects of HG on the level of phospho-p38MAPKTyr180/Tyr182 and phospho-STAT1Ser727. (e). In VEGF knockdown cell (siVEGF, 20 nM), apoptotic cell death was determined following HG treatment. (f). Cells were grown on Transwell filters, and VEGF was knocked down using siVEGF (20 nM). Following HG treatment, the permeability of monolayer cells was measured. Data are presented mean ± SD. a, c and d: n = 3; B: n = 4; e and f: n = 5. *p < 0.05 vs CTL, #p < 0.05 vs HG or HG + siCTL; n.s. non-significance. In c and d, stripped membranes from phospho-Erk, phospho-p38MAPK, and phospho-STAT1 were reblotted for total Erk, p38MAPK, and STAT1, respectively.
Fig 3: The expression of selected PSD-related proteins in forebrains of AgNPs/Ag citrate-exposed immature rats early (PND 35) after exposure. Representative immunoblots and graphs indicating relative expression of respective proteins and their mRNAs: PSD95 (A,B), SynGAP (C,D) and nNOS (E,F). Data are means ± SD from four independent experiments; * p < 0.05, ** p < 0.01 vs. control, # p < 0.05 vs. AgNPs (one-way ANOVA followed by Tukey’s multiple comparison test).
Fig 4: Immunocytochemical localization and expression of nNOS and iNOS in the spinal cord of tumor-bearing mice. (A) nNOS immunostaining in the lumbar spinal cord at day 10 after tumor-cell inoculation. (C) iNOS immunostaining in the lumbar spinal cord at day 14 after tumor-cell inoculation. Magnification, x40. The nNOS and iNOS immunoreactive cells (arrows) were mainly located in the dorsal horn of the spinal cord (laminae I-II) and a few in the ventral parts and around the central canal. (B) nNOS and (D) iNOS immunostaining in the IPSI side of the superficial dorsal horn at higher magnification, ×200. Statistical analysis of mean optical density of (E) nNOS and (F) iNOS. Values are expressed as the mean ± standard deviation. *P<0.05 vs. sham group at the same time-point, #P<0.05 vs. the same group of mice at day 7. IPSI, ipsilateral; iNOS, inducible nitric oxide synthase; nNOS, neuronal NOS.
Fig 5: The expression of selected PSD-related proteins in forebrains of AgNPs/Ag citrate-exposed immature rats late (PND 90) after exposure. Representative immunoblots and graphs indicating relative expression of respective proteins and their mRNAs: PSD95 (A,B), SynGAP (C,D) and nNOS (E,F). Data are means ± SD from four independent experiments; * p < 0.05, ** p < 0.01 vs. control (one-way ANOVA followed by Tukey’s multiple comparison test).
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