Journal: Cell reports

Article Title: Interaction of RAGE with α-synuclein fibrils mediates inflammatory response of microglia.

doi: 10.1016/j.celrep.2022.111401

Figure Lengend Snippet: Figure 2. C-terminal of a-syn plays a major role in the binding with vRAGE (A) Overlay of the 2D 1H-15N HSQC spectra of a-syn monomer alone (black) and that titrated by vRAGE (blue) at a molar ratio of 1:2 (a-syn:vRAGE). The cross- peaks of the negatively charged residues with significant CSDs (CSDs > 0.01 ppm) at the C terminus of a-syn are highlighted with black boxes and zoomed in on the top right. Experiments were performed two independent times. (B) Residue-specific CSDs of a-syn monomer titrated by vRAGE from (A). The domain organization of a-syn is shown on the top. The primary sequence of a-syn C terminus (residues 101–140) is shown, and the acidic residues are colored in red. The residues with CSDs >0.01 ppm are highlighted in blue. (C) NS-TEM image of a-syn1-100 fibrils. Scale bar, 100 nm. Experiments were performed three independent times. (D) The binding kinetics of vRAGE with a-syn1-100 monomer (left) and a-syn1-100 PFF (right) determined by BLI assay. Experiments were performed in triplicate. Data are processed with GraphPad Prism 7. N.D., not detected.

Article Snippet: The plates were colorated with 1-StepTM NBT/BCIP Substrate Solution (ThermoFisher, 34042) at RT for 10 min. And cells were imaged with the Leica DFC365 FX fluorescence microscopy and analyzed with Image J. Quantitative polymerase chain reaction BV2 cells were plated and treated with PBS, LPS (100 ng/mL), a-syn monomer (5 mM), a-syn PFFs (5 mM), a-syn1-100 PFFs (5 mM) and FPS-ZM1 (MCE, 945714-67-0) for 7 h, respectively.

Techniques: Binding Assay, Residue, Sequencing

Journal: Cell reports

Article Title: Interaction of RAGE with α-synuclein fibrils mediates inflammatory response of microglia.

doi: 10.1016/j.celrep.2022.111401

Figure Lengend Snippet: Figure 5. a-Syn PFFs increase the expression of cytokines dependent on RAGE (A) Statistical analysis of the mRNA fold change of TNF-a, IL-1b, and IL-6. BV2 cells were treated with PBS (gray), LPS (red), a-syn monomer (pink), a-syn WT PFFs (dark blue), and a-syn1-100 PFFs (light blue), respectively. (B) Statistical analysis of mRNA fold change of TNF-a, IL-1b, and IL-6 after BV2 cells treated with PBS (gray), a-syn WT PFFs (dark blue), and a-syn WT PFFs in the presence of FPS-ZM1 at molar ratios of 1:1 (light green) and 1:5 (dark green). (C) Statistical analysis of mRNA fold change of TNF-a, IL-1b, and IL-6 after primary microglia cells treated with a-syn monomer (pink), a-syn WT PFFs (dark blue), and a-syn WT PFFs in the presence of FPS-ZM1 at molar ratios of 1:1 (light green) and 1:5 (dark green). (D) Statistical analysis of mRNA fold change of TNF-a, IL-1b, and IL-6. WT (solid) and RAGE knockout (KO) (texture) primary microglia were treated with a-syn monomer (pink) and a-syn WT PFFs (dark blue), respectively. The data were analyzed with GraphPad Prism 7. Data are the means ± SD; all experiments were performed in duplicate and performed at least three times. One-way ANOVA followed by Tukey’s correction. ns, p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Statistical information is provided in STAR Methods. See also Figure S5.

Article Snippet: The plates were colorated with 1-StepTM NBT/BCIP Substrate Solution (ThermoFisher, 34042) at RT for 10 min. And cells were imaged with the Leica DFC365 FX fluorescence microscopy and analyzed with Image J. Quantitative polymerase chain reaction BV2 cells were plated and treated with PBS, LPS (100 ng/mL), a-syn monomer (5 mM), a-syn PFFs (5 mM), a-syn1-100 PFFs (5 mM) and FPS-ZM1 (MCE, 945714-67-0) for 7 h, respectively.

Techniques: Expressing, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 2. DM mice exhibit synaptic deficits and increased neuroinflammation in the HIP. (A) Repre- sentative WB images showing the expression levels of the presynaptic protein synapsin I (SYN1) and the postsynaptic protein postsynaptic density protein 95 (PSD95) in the HIP of CTL and DM mice. β-actin was used as an internal control. (B) Semi-quantitative analysis of PSD95 and SYN1 expression levels from immunoblot experiments. n = 6. (C) Representative images of Iba1 immunostaining in the HIP of CTL and DM mice. The area within the dashed box is magnified and displayed in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (D) Quantitative analysis of Iba1-positive microglia in the HIP of CTL and DM mice. n = 5. (E) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of CTL and DM mice. n = 6. (F) qPCR analysis of IL-6, IL-1β, and TNF-α mRNA levels in the HIP of CTL and DM mice. n = 6. (G) ELISA measuring IL-6, IL-1β, and TNF-α levels in the serum of CTL and DM mice. n = 3. Data are presented as mean ± SEM. Statistical significance was determined using a one-tailed unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Expressing, Control, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mesenchymal Stem Cells Restore Endothelial Integrity and Alleviate Emotional Impairments in a Diabetic Mouse Model via Inhibition of MMP-9 Activity

doi: 10.3390/ijms26073355

Figure Lengend Snippet: Figure 6. MSC treatment attenuated DM-induced neuroinflammation and synaptic deficits. (A) Representative immunofluorescence images of Iba1 staining in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. The dashed box indicates the magnified region shown in the insets. Scale bars: 100 µm (main images) and 20 µm (insets). (B) Quantitative analysis of Iba1+ microglia in the HIP. n = 4. (C) qPCR analysis of iNOS and Arg1 mRNA levels in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (D) qPCR analysis of mRNA levels of IL-6, IL-1β, and TNF-α in the HIP of mice from the CTL, MSC, DM, and DM+MSC groups. n = 9. (E) ELISA results showing serum levels of IL-6, IL-1β, and TNF-α in CTL, MSC, DM, and DM+MSC mice. n = 4. (F) Representative WB images of PSD95 and SYN1 expression in the HIP. β-actin was used as a loading control. (G) Semi-quantitative analysis of PSD95 and SYN1 protein levels. n = 6. Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked and incubated overnight with the following antibodies: PSD95 (SYSN, Toronto, ON, Canada, 124002, 1:1000); SYN1 (SYSN, 106011, 1:1000); Cldn5 (Invitrogen, 35-2500, 1:1000); Ocln (Invitrogen, 71-1500, 1:1000); and MMP9 (Boster, Shanghai, China; PB9669, 1:1000).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Quercetin Alleviates LPS-Induced Depression-Like Behavior in Rats via Regulating BDNF-Related Imbalance of Copine 6 and TREM1/2 in the Hippocampus and PFC

doi: 10.3389/fphar.2019.01544

Figure Lengend Snippet: Effect of quercetin on the protein expression of brain derived neurotrophic factor (BDNF), Copine 6, TREM1, TREM2, and Synapsin-1 in the hippocampus and prefrontal cortex (PFC) of lipopolysaccharide (LPS)-challenged rats (A) shows a typical graph, and (B) and (C) show the statistical analysis of western blotting results. The data in (B) and (C) are presented as the mean ± SEM, with n = 3 for each group. The protein expression of BDNF, Copine 6, and TREM1 were decreased in both the hippocampus and the PFC of LPS-challenged rats, while the expression of TREM2 were increased, and the protein expression of synapsin-1 were decreased in the hippocampus without significant changes in the PFC. These changes could be balanced by treatment of ibuprofen, fluoxetine, and quercetin. * P < 0.05 and ** P < 0.001, compared with the control group; # P < 0.05 and ## P < 0.001, compared with the NAFLD model group. 1 control; 2 Model; 3 Ibuprofen; 4 Fluoxetine; 5 Quercetin.

Article Snippet: The membrane was blocked with 5% skim milk for 1 h; incubated with antibodies targeting BDNF (1:1000; Abcam, Cambridge, UK), TrkB (1:10000; Abcam, Cambridge, UK), phosphorylated TrkB (p-TrkB) (1:10000; Abcam, Cambridge, UK), Copine 6 (1:1,000; Santa Cruz Biotechnology, Inc., Delaware, USA), synapsin-1 (1:1000; ImmunoWay, Newark, Delaware, USA), TREM-1 (1:1000; ImmunoWay, Newark, Delaware, USA), TREM-2 (1:1000; ImmunoWay, Newark, Delaware, USA), or β-actin (1:1000; Zhongshan Biotechnology, INC, Beijing, China) at 4°C overnight, and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2000) at 37°C for 2 h. The blots were developed with the Easy Enhanced Chemiluminescence Western Blot Kit (Pierce Biotechnology, Rockford, IL, USA).

Techniques: Expressing, Derivative Assay, Western Blot