Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Inhibition of fatty acid‐binding protein (A‐FABP) in microglia attenuates neuronal apoptosis and blood–brain barrier disruption. (A and B) Representative western blot image and quantitative analysis of A‐FABP expression at 0, 12 and 24 h post‐PA (200 µM) treatment ( n = 6). (C and D) Representative western blot image and quantitative analysis of A‐FABP expression at 0, 12 and 24 h post‐AA (100 µM) treatment ( n = 6). (E) Schematic diagram of microglial culture supernatant transfer model (PA, 200 µM; BMS, 20 µM). (F and G) Representative flow cytometry images of primary neuron cells apoptosis in PA‐induced microglial culture supernatant transfer model ( n = 6). (H) Schematic representation of bEnd.3 endothelial cell co‐culture system with BV2 microglial conditioned medium (CM) treated with PA (200 µM), with or without BMS (20 µM). (I and J) Representative microphotographs of immunofluorescence and quantitative analysis in bEnd.3 cells co‐cultured with the CM of BV2 cells (scale bar = 100 µm, n = 6). Data are presented as means ± SD. * p < .05, ** p < .01, **** p < .0001.

Article Snippet: To mimic the fatty acid model in vitro, BV2 cells were treated with AA (#HY‐109590, 100 μM; MCE) and palmitic acid (PA, #P0500, 200 μM; Sigma‐Aldrich) as previously described.

Techniques: Inhibition, Binding Assay, Disruption, Western Blot, Expressing, Flow Cytometry, Co-Culture Assay, Immunofluorescence, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Fatty acid‐binding protein (A‐FABP) suppression promotes fatty acid β‐oxidation reprogramming in microglia. (A) Schematic illustration of experimental design. Control group: PBS treatment. FFA group: 200 µM palmitic acid (PA). BMS group: Co‐treatment with PA (200 µM) and BMS (20 µM). (B–D) Oxygen consumption rate (OCR) was determined in BV2 cells treated with PA, with or without BMS ( n = 6). Quantitative analyses of (C) basal respiration and (D) maximal respiration. (E–H) Glycolytic proton efflux rate (GlycoPER) was determined in BV2 cells treated with PA, with or without BMS ( n = 6). Quantitative analyses of (F) basal glycolysis, (G) basal proton efflux rate and (H) compensatory glycolysis. (I–K) Representative western blot images and quantitative analyses of (J) CPT1A and (K) ACADL in different groups ( n = 6). (L) ATP content assay in different groups ( n = 6). Data are presented as means ± SD. * p < .05, **** p < .0001. glycoPER, glycolytic proton efflux rate. Source : Schematic diagram created with BioRender.com.

Article Snippet: To mimic the fatty acid model in vitro, BV2 cells were treated with AA (#HY‐109590, 100 μM; MCE) and palmitic acid (PA, #P0500, 200 μM; Sigma‐Aldrich) as previously described.

Techniques: Binding Assay, Control, Western Blot

Journal: Global Spine Journal

Article Title: miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1

doi: 10.1177/21925682251406197

Figure Lengend Snippet: The Expression of miR-369-3p was Down-Regulated in the SCI Mice Model. (A) The Expression of miR-369-3p in the Spinal Cord Tiss ue of SCI Mice was Detected by RT-qPCR. (B) The Expression of miR-369-3p in BV2 Microglia Induced by Different Concentrations of LPS was Detected by RT-qPCR. n = 6 Per Group; ** P < 0.01, *** P < 0.001

Article Snippet: BV2 microglia cells (Cat# CRL-2469, ATCC, USA) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Cat#26140, Invitrogen, USA) and 1% penicillin-streptomycin (Cat#15140, Invitrogen, USA) at 37°C in a 5% CO 2 and humidity over 95%.

Techniques: Expressing, Quantitative RT-PCR

Journal: Global Spine Journal

Article Title: miR-369-3p Regulates Microglia Polarization and Neuroinflammation in Traumatic Spinal Cord Injury by Targeting PELI1

doi: 10.1177/21925682251406197

Figure Lengend Snippet: miR-369-3p Inhibits LPS-Induced M1 Polarization of Microglia and the Expression of Inflammatory Factors. A. Effect of miR-369-3p Mimic Transfection on the Expression of miR-369-3p in LPS-Induced BV2 Cells. B-D. The Expression of M1 Polarization Markers CD86 and iNOS, and M2 Markers Arg-1 mRNA and Protein Levels in Response to the Combined Effects of LPS and miR-369-3p Mimic. E-F. The mRNA and Concentration of the Inflammation Factor TNF-α, IL-6, and IL-1β in the LPS-Induced BV2 Cells. n = 6 Per Group; *** P < 0.001 vs Control; ### P < 0.001 vs LPS + NC Mimic

Article Snippet: BV2 microglia cells (Cat# CRL-2469, ATCC, USA) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Cat#26140, Invitrogen, USA) and 1% penicillin-streptomycin (Cat#15140, Invitrogen, USA) at 37°C in a 5% CO 2 and humidity over 95%.

Techniques: Expressing, Transfection, Concentration Assay, Control

Journal: Journal of Biomedical Research

Article Title: HDGF derived from Müller cells enhances the activation of microglia in diabetic retinopathy

doi: 10.7555/JBR.38.20240386

Figure Lengend Snippet: HDGF promoted microglia activation and IL-1β secretion. A: Analysis of a published list of target genes of HDGF integrated with mouse retinal single-cell RNA sequencing data, indicating predominant target cell types. The violin plot shows expression of the target gene Itgb2 in microglia from control and streptozotocin (STZ)-induced diabetic mouse retinas. The microglia cluster is indicated. B: Enzyme-linked immunosorbent assay measuring IL-1β secretion in the supernatant of BV2 microglial cells. Cells were treated under low-glucose (LG, 1 g/L) or high-glucose (HG, 5 g/L) conditions for 48 h, with or without recombinant HDGF (rHDGF) and an HDGF-neutralizing antibody (anti-HDGF) ( n = 3). C: Quantitative PCR analysis of Itgb2 mRNA expression in BV2 cells treated as indicated ( n = 3). D: Representative immunofluorescence images (left) and quantification (right) of ITGB2-positive BV2 cells following the treatments. Nuclei were counterstained with DAPI. Quantitative analysis of ITGB2-positive cells treated as indicated ( n = 3). E: Representative immunofluorescence images (left) and quantification (right) of CD68-positive BV2 cells following the treatments. Nuclei were counterstained with DAPI. Quantitative analysis of CD68-positive cells treated as indicated ( n = 3). F: Feature plot from retinal scRNA-seq data showing specific Itgb2 expression within the microglia cluster (outlined). G: Western blotting analysis of ITGB2 protein expression in BV2 microglial cells and primary Müller cells. α-Tubulin served as the loading control. Quantitative protein levels of ITGB2 in BV2 and Müller cells ( n = 3 per group). Data are presented as mean ± standard deviation. Statistical significance was determined by one-way analysis of variance (ANOVA; B–E) or paired Student's t -test (G). * P < 0.05; ** P < 0.01; *** P < 0.001. Abbreviations: HDGF, hepatoma-derived growth factor; ITGB2, integrin beta 2.

Article Snippet: The immortalized murine microglial cell line (BV2) (Cat. #CRL-2469) was purchased from the American Type Culture Collection and cultured in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Invitrogen), 1% glutamine (Invitrogen), and streptomycin (100 U/mL; Invitrogen) at 37 °C, 5% CO 2 .

Techniques: Activation Assay, RNA Sequencing, Expressing, Control, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Immunofluorescence, Western Blot, Standard Deviation, Derivative Assay

Journal: Journal of Biomedical Research

Article Title: HDGF derived from Müller cells enhances the activation of microglia in diabetic retinopathy

doi: 10.7555/JBR.38.20240386

Figure Lengend Snippet: High glucose upregulated the expression of HDGF receptor in microglia. A: Western blotting analysis of HDGF protein expression in BV2 microglial cells cultured under low-glucose (LG, 1 g/L) or high-glucose (HG, 5 g/L) conditions for 48 h. α-Tubulin served as the loading control. Quantitative protein levels of ITGB2 in BV2 and Müller cells ( n = 3 per group). B: Representative immunofluorescence images of retinal cryosections from control and streptozotocin (STZ)-induced diabetic mice, co-stained for HDGF (red) and the microglial marker IBA1 (green). Nuclei were counterstained with DAPI (blue). Assessment of immunofluorescence colocalization of HDGF and IBA1 in the two experimental groups (left, control; right, STZ). C: Single-cell RNA sequencing analysis of control and STZ-diabetic mouse retinas. UMAP plot showing the expression of Ncl (the gene encoding nucleolin) across all cells (upper). The microglia cluster is indicated. Dot plot illustrating the relative expression level and proportion of Ncl -expressing cells in microglia in the control and STZ groups (lower). D: Representative immunofluorescence images (left) and quantification of mean fluorescence intensity (MFI, right) for NCL (green) in BV2 cells cultured under LG or HG conditions for 48 h. Nuclei were counterstained with DAPI (blue). n = 3. Data are presented as mean ± standard deviation. Statistical significance was determined by the paired Student's t -test. * P < 0.05. Abbreviations: HDGF, hepatoma-derived growth factor; IBA1, ionized calcium-binding adaptor molecule 1; ns, not significant; STZ, streptozotocin.

Article Snippet: The immortalized murine microglial cell line (BV2) (Cat. #CRL-2469) was purchased from the American Type Culture Collection and cultured in DMEM/F12 medium (Gibco) supplemented with 10% FBS (Invitrogen), 1% glutamine (Invitrogen), and streptomycin (100 U/mL; Invitrogen) at 37 °C, 5% CO 2 .

Techniques: Expressing, Western Blot, Cell Culture, Control, Immunofluorescence, Staining, Marker, RNA Sequencing, Fluorescence, Standard Deviation, Derivative Assay, Binding Assay