Journal: International Journal of Molecular Sciences

Article Title: Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion

doi: 10.3390/ijms21062176

Figure Lengend Snippet: Increased high-mobility group box protein 1 (HMGB1) and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α] and interleukin [IL]-1β) in the cortex and hippocampus at 3 months after chronic cerebral hypoperfusion (CCH). ( A ) Western blot results of HMGB1 in the cortex and hippocampus. ( B ) Quantification results of HMGB1 relative protein in the cortex and hippocampus. ( C ) Immunostaining of HMGB1, TNF-α, and IL-1β in the cortex. ( D ) Quantification data of immunostaining image. n = 6 for each group. Data are presented as the mean + standard deviation; * p < 0.05; ** p < 0.01 with unpaired t-test; scale bar = 50 μm.

Article Snippet: Equal amounts of protein (20 μg) were then separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a primary Ab against HMGB1 (mouse monoclonal Ab, 1:1000, GeneTex, Irvine, CA, USA), interleukin (IL)-1α (rabbit polyclonal Ab, 1:1000, GeneTex, Irvine, CA, USA), tumor necrosis factor-alpha (TNF-α; rabbit polyclonal Ab, 1:1000, GeneTex, USA), and IL-6 (rabbit polyclonal Ab, 1:1000, GeneTex, Irvine, CA, USA).

Techniques: Western Blot, Immunostaining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion

doi: 10.3390/ijms21062176

Figure Lengend Snippet: The effect of high-mobility group box protein 1 (HMGB1) suppression with CRISPR/Cas9 knockout (KO) plasmid on proinflammatory cytokines, hippocampus volume, and memory function. ( A ) Decreased level of tumor necrosis factor-alpha, interleukin (IL)-1β, and IL-6 of bilateral common carotid artery occlusion (BCCAO) HMGB1-KO mice showed with immunostaining. ( B ) Attenuation of hippocampal atrophy in BCCAO HMGB1-KO mice but not in BCCAO controls (the area of the hippocampus is indicated with a red circle). ( C ) Improvement of memory decline in BCCAO HMGB1-KO mice but not in BCCAO controls. n = 5 for each group. Data are presented as the mean + standard deviation; * p < 0.05; ** p < 0.01; *** p < 0.001 with one-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison test; novel object recognition test before and after CRISPR/Cas9 injection with two-way ANOVA (F(3,24) = 7.598, p < 0.01); scale bar = 100 μm.

Article Snippet: Equal amounts of protein (20 μg) were then separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a primary Ab against HMGB1 (mouse monoclonal Ab, 1:1000, GeneTex, Irvine, CA, USA), interleukin (IL)-1α (rabbit polyclonal Ab, 1:1000, GeneTex, Irvine, CA, USA), tumor necrosis factor-alpha (TNF-α; rabbit polyclonal Ab, 1:1000, GeneTex, USA), and IL-6 (rabbit polyclonal Ab, 1:1000, GeneTex, Irvine, CA, USA).

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Immunostaining, Standard Deviation, Comparison, Injection

Journal: PLoS ONE

Article Title: A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

doi: 10.1371/journal.pone.0083119

Figure Lengend Snippet: List of primary antibodies used for immunofluorescence (IF) and western blot (WB) studies.

Article Snippet: The cells were detached with trypsin, washed in PBS, pelleted at 300 g for 10 min and fixed with PFA 2% for 20 min. Then, cells were permeabilized with 0,2% Triton in PBS for 20 min, blocked with 10% NGS in PBS for half hour, and incubated with primary antibody TNF-α (1:100; MBL) for 1 hour in blocking solution.

Techniques: Immunofluorescence, Western Blot, Binding Assay, Purification

Journal: PLoS ONE

Article Title: A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

doi: 10.1371/journal.pone.0083119

Figure Lengend Snippet: A) After 7 DIV organotypic cultures were treated with TFM-C (50µM) for 6h and then stimulated with 15µg/ml LPS for 24h in presence of TFM-C. Panel a) Lay-out of cytokine-specific antibody spots in the 16-plex cytokine Stripwell array (left image) and visualization of cytokine-specific chemiluminescence in culture medium of LPS-treated organotypic cultures in the absence or presence of TFM-C (right images). Cyotkine levels significantly affected by TFM-C are highlighted in grey. Panel b) pg/ml of cytokines were indicated. sd: standard deviation. ND: not determinable. B) Effect of TFM-C on IL-1β, IL-6, IL-10, IL-12p35, IL-23p19, TNF-α and HERP mRNA in organotypic cultures stimulated by LPS for 6h and 24h in presence or absence of 50µM TFM-C. The levels of mRNA are shown as n -fold increase compared with baseline level (-) and normalized to those of the housekeeping gene Hprt1 . Asterisks indicate significant differences at *< P 0.05 compared with LPS control by ANOVA test.

Article Snippet: The cells were detached with trypsin, washed in PBS, pelleted at 300 g for 10 min and fixed with PFA 2% for 20 min. Then, cells were permeabilized with 0,2% Triton in PBS for 20 min, blocked with 10% NGS in PBS for half hour, and incubated with primary antibody TNF-α (1:100; MBL) for 1 hour in blocking solution.

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

doi: 10.1371/journal.pone.0083119

Figure Lengend Snippet: BV2 cells were treated with TFM-C (30 or 50µM) for 2h and then stimulated with LPS (1µg/ml) for different times in presence or absence of TFM-C. A) Quantification of the kinetics of mRNA production and cytokine secretion (IL-1β, IL-6 and TNF-α). All values represent the averages of three independent experiments. Lower graphs represent cytokine-specific mRNA quantified by QPCR, while the upper graphs represents amount of secreted cytokine quantified using specific ELISA kits. Asterisks indicate significant differences at * P < 0.05, ** P <0.01, *** P <0.001 between TFM-C-treated and LPS-treated cells at each time point using ANOVA test. B) Effect of TFM-C (30 and 50µM) on IL-23p19 and HERP mRNAs in BV2 cells stimulated with LPS. The levels of mRNA levels are shown as fold increase. Asterisks indicate significant differences at * P < 0.05 and ** P <0.01 compared with LPS only using ANOVA test. C) Effect of TFM-C on HERP protein expression. BV2 cells were treated with TFM-C (50µM) or pretreated with TFM-C for 2h (30 and 50µM) and then stimulated with LPS in presence of TFM-C. Panel a) Immunofluorescence for HERP (green), Iba1 (red) and DAPI (blue) at 12h of LPS/TFM-C or TFM-C treatment. Scale bar 5µm. Panel b-c) 10µg of total protein were loaded for HERP Western blot analysis. Results were expressed as arbitrary units respect to the control at same time point. Error bars indicate the standard error. * P <0.05 by ANOVA test. D) Effect of TFM-C treatment (50µM) on the viability of BV2 cells. Apoptotic cells were measured by double propidium iodite (PI) and DAPI staining, and the percentage of damaged DNA and condensed chromatin was calculated at 24h of LPS/TFM-C treatment.

Article Snippet: The cells were detached with trypsin, washed in PBS, pelleted at 300 g for 10 min and fixed with PFA 2% for 20 min. Then, cells were permeabilized with 0,2% Triton in PBS for 20 min, blocked with 10% NGS in PBS for half hour, and incubated with primary antibody TNF-α (1:100; MBL) for 1 hour in blocking solution.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Western Blot, Staining

Journal: PLoS ONE

Article Title: A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

doi: 10.1371/journal.pone.0083119

Figure Lengend Snippet: A) BV2 cells were treated with TFM-C (50µM) for 2h and stimulated with LPS (1µg/ml) for 3, 6 and 24h in presence or absence of TFM-C. Panel a) Staining for TNF-α in permeabilized cells. Scale bar 5µm. Panel b) Intracellular localization of TNF-α by flow cytometry. Results are expressed as fold increase compared to the control at the same time point. B) TNF-α release in astrocyte cultures. Astrocytes were treated with TMF-C (30 or 50µM) for 24h or pre-treated with TMF-C for 2h and then stimulated with LPS (1µg/ml) in presence or absence of TFM-C for 24h and then analyzed by ELISA. Error bars indicate the standard deviation. * P <0.05, ** P <0.01, *** P <0.001 by ANOVA test.

Article Snippet: The cells were detached with trypsin, washed in PBS, pelleted at 300 g for 10 min and fixed with PFA 2% for 20 min. Then, cells were permeabilized with 0,2% Triton in PBS for 20 min, blocked with 10% NGS in PBS for half hour, and incubated with primary antibody TNF-α (1:100; MBL) for 1 hour in blocking solution.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLoS ONE

Article Title: A Trifluoromethyl Analogue of Celecoxib Exerts Beneficial Effects in Neuroinflammation

doi: 10.1371/journal.pone.0083119

Figure Lengend Snippet: BMDCs were incubated in TFM-C, celecoxib or vehicle for 16h and subsequently stimulated with LPS (0.1µg/ml) (A) or heat killed H37Ra Mtb (10µg/ml) (B) in the presence of TFM-C, celecoxib or vehicle. Cytokines were detected by ELISA. IL-12, IL-1β or IL-6 were measured 24h after stimulation. IL-23 and TNF-α were measured 6h after stimulation. The data shown are pooled from two similar experiments. Error bars represent + SEM. * P <0.05 control versus TFM-C celecoxib-treated group, ** P <0.05 control versus TFM-C -treated group.

Article Snippet: The cells were detached with trypsin, washed in PBS, pelleted at 300 g for 10 min and fixed with PFA 2% for 20 min. Then, cells were permeabilized with 0,2% Triton in PBS for 20 min, blocked with 10% NGS in PBS for half hour, and incubated with primary antibody TNF-α (1:100; MBL) for 1 hour in blocking solution.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay