Journal: Stroke and vascular neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage.

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Figure 3 Human serum amyloid A1 (SAA1) protein levels in plasma and extracellular vesicles correlated (EVs) with clinical and laboratory assessments. (A, B) Bar graph indicating the concentration of SAA1 protein in both EVs and plasma for the ICH group and the healthy controls group. Unit: ng/mL in EVs and μg/mL in plasma. **p<0.01, ***p<0.001 by the two-tailed unpaired Student’s t-test and data are presented as means±SEM. (A) n=6, 15 in the control and ICH group, (B) n=13, 19 in the control and the ICH group. (C, D) Linear dependence graph revealing the relationship between neutrophils or leucocytes and the SAA1 protein in EVs (n=15). Correlation was analysed by spearman correlation analysis and the dashed line indicates the 95% CI. (E–H) Correlation analysis between the EVs-derived, plasma-derived SAA1 levels and the clinical assessments, including NIHSS and haemorrhage volume, through a linear dependence graph (n=15). Correlation was analysed by spearman correlation analysis and the dashed line indicates the 95% CI. ICH, intracerebral haemorrhage; NIHSS, National Institutes of Health Stroke Scale.

Article Snippet: We used anti- mouse SAA1 mAb (AF2948, NOVUS, USA) to block SAA1 in vivo at a dose of 0.25 μg/g, as previously reported.24 To establish a control, we administered Goat IgG Isotype Control (AB- 108- C, NOVUS, USA).

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test, Control, Derivative Assay

Journal: Stroke and vascular neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage.

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Figure 4 Exogenous SAA1 upregulates the cell counts of microglia and astrocytes. (A) Schematic representation of exogenous SAA1 inserting into the basal ganglia region at varying dosages (750 ng, 250 ng, 83 ng and 0 ng). Images of immunostaining of microglia and astrocyte activation on day three are provided. (B, D) Images of immunostaining (B) and quantification (D) of microglia (stained by a specific marker, Iba1) around the ipsilateral basal ganglia region at different doses of exogenous SAA1. Microglia counts per mm2: 9.3±2.2, 41.5±3.9, 64.0±4.0, 88.3±8.8 for 0 ng, 83 ng, 250 ng, and 750 ng, respectively. (C, E) Images of immunostaining of GFAP (glial fibrillar acidic protein, known as the astrocyte active marker) representing astrocytes (C) and quantification of astrocytes (E) at different doses of exogenous SAA1 in the ipsilateral basal ganglia region. Astrocyte counts per mm2: 36.0±3.8, 93.5±6.1, 134.0±3.5, 176.5±11.6 for 0 ng, 83 ng, 250 ng, and 750 ng, respectively. Scale bars=100 µm, inset scale bars=20 µm, n=4 in each group. ***p<0.001 by the Kruskal-Wallis test. Data are presented as means±SEM.

Article Snippet: We used anti- mouse SAA1 mAb (AF2948, NOVUS, USA) to block SAA1 in vivo at a dose of 0.25 μg/g, as previously reported.24 To establish a control, we administered Goat IgG Isotype Control (AB- 108- C, NOVUS, USA).

Techniques: Immunostaining, Activation Assay, Staining, Marker

Journal: Stroke and vascular neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage.

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Figure 5 Blocking SAA1 promotes microglia reactivity and leucocyte infiltration. (A) Bar graph indicating the elevation of plasma SAA1 levels in ICH mice as compared with the sham group. n= 5 mice in the ICH group and n=10 mice in the sham group. **p<0.01 by two-tailed unpaired Student’s t-test. (B) Schematic diagram depicting intracerebral haemorrhage induction followed by intravenous administration of anti-SAA1 mAb or IgG 1 hour later. Immune cell populations were assessed using flow cytometry on days 1 and 3 post-ICH initiation. (C) Flow cytometry gating strategy depicting immune cell populations in mice brain treated with anti-SAA1 antibody or IgG on days 1 and day three post-ICH induction. The graph illustrates CD45high leucocytes, including CD3+CD19- T lymphocytes and its subtypes: CD4+CD8− T and CD4-CD8+ T lymphocytes, CD3−CD19+ B lymphocytes, CD11b+Ly6G+ neutrophils, and CD11b+F4/80+ macrophages. It also illustrates CD45int CD11b+microglia, including its subtypes: CD86+ microglia and CD206+ microglia. All gates were set using fluorescence-minus-one (FMO) controls. (D) Bar graph indicates the number of microglia and their subtypes in ICH mice with anti-SAA1 antibody or IgG treatment from days 1 to 3. (E) Bar graph shows major brain infiltrated leucocytes, involving CD8+ T lymphocytes, B lymphocytes, and neutrophils in ICH mice with anti-SAA1 antibody or IgG treatment from days 1 to 3. Int, intermediate. n=5, 6, 5 on day 1 and n=5, 13, 10 on day 3 for sham, IgG and mAb group. n=5, 9, 7 on day 3 for CD86+ microglia group in sham, IgG and mAb group specially. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA and Tukey’s test. Data are presented as means±SEM. ANOVA, analysis of variance; ICH, intracerebral haemorrhage.

Article Snippet: We used anti- mouse SAA1 mAb (AF2948, NOVUS, USA) to block SAA1 in vivo at a dose of 0.25 μg/g, as previously reported.24 To establish a control, we administered Goat IgG Isotype Control (AB- 108- C, NOVUS, USA).

Techniques: Blocking Assay, Clinical Proteomics, Two Tailed Test, Flow Cytometry, Fluorescence

Journal: Stroke and vascular neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage.

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Figure 6 Anti-SAA1 mAb administration alleviates brain injury in mice with ICH. (A) Schematic diagram illustrating the ICH followed by intravenous injection of anti-SAA1 mAb or IgG 1 hour later. Subsequently, mice underwent MRI and neurological evaluations on days 1 and 3 after ICH induction. (B) Neurological scores of the sham group and the ICH group treated with anti- SAA1 mAb or IgG on day 1 and day 3. The modified Neurological Severity Score (mNSS) and rotarod test were used to measure the neurological deficit. n=6, 12, 7 for sham, IgG and mAb group. *p<0.05 by two-way ANOVA. (C) MRI of lesion volume (red) and perihaemorrhagic oedema (PHE) volume (yellow) on day 1 and day 3 post-ICH. (D, E) Quantification of lesion volume and PHE volume using the MRI. n=4, 6 for IgG and mAb group on day 1, n=5, 8 for IgG and mAb group on day 3. *p<0.05 by two-tailed unpaired Student’s t-test. Data are presented as means±SEM. ANOVA, analysis of variance; ICH, intracerebral haemorrhage.

Article Snippet: We used anti- mouse SAA1 mAb (AF2948, NOVUS, USA) to block SAA1 in vivo at a dose of 0.25 μg/g, as previously reported.24 To establish a control, we administered Goat IgG Isotype Control (AB- 108- C, NOVUS, USA).

Techniques: Injection, Modification, Two Tailed Test

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Glomerular parietal epithelial cell activation induces collagen secretion and thickening of Bowman's capsule in diabetes.

doi: 10.1038/labinvest.2014.160

Figure Lengend Snippet: Figure 2 Metabolic alterations in DN activate PECs as displayed by CD44 expression and changes in the ultrastructural morphology of the PEC nuclei. On PECs, CD44 is expressed at late phases of activation and associated with a thickened Bowman’s capsule in a patient with stage IV DN (a) in comparison with a patient in the non-diabetic control group, where PECs do not express CD44 (b) under 400 magnification. Ultrastructural activation of PECs is displayed as enlarged diameter of the nuclei (43.5 mm) and euchromatic nuclei assessed by TEM under 1600 magnification of a patient with stage IIa DN (d) in comparison with a patient in the non-diabetic control group (c). DN, diabetic nephropathy; PECs, parietal epithelial cells; TEM, transmission electron microscopy.

Article Snippet: Sections were then incubated with anti-CD44 antibody (1:100, mouse monoclonal Ab 156-3C11; Novus Biologicals, Littleton, CO, USA) for 60 min at room temperature.

Techniques: Expressing, Activation Assay, Comparison, Control, Transmission Assay, Electron Microscopy

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 1. Isolation of AF9-binding proteins. THP-1 whole cell extract was pre-adsorbed to a FLAG peptide affinity matrix and was then applied to a FLAG-AF9-ct column (amino acids 475–568 of AF9). Bound proteins were eluted with 1 M NaCl and 100 mM glycine. Eluted proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Bands 1–6 were excised from the gel and identified by mass spectroscopy. The 85-kDa band is the isoform of Hsp90. The gel shown is the original and only replicate.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: Isolation, Binding Assay, SDS Page, Staining, Mass Spectrometry

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 2. In vitro binding of AF9 and Hsp90. A, purified recombinant FLAG-AF9-ct and Hsp90 were used to determine if the proteins interacted directly and in the absence of other factors. FLAG peptide was included as a control. Proteins were incubated in the presence or absence of the non-hydrolyzable ATP analog ATPS. Proteins were then recovered by anti-FLAG immunoprecipitation and analyzed by Western blot for Hsp90. The Table indicates the composition of the incubation mixture. Hsp90 co-precipitates with FLAG-AF9-ct but only when ATP is present. The figure is representative of three experiments. B, competitive ELISA was used to determine the Kd of the Hsp90 and AF9 interaction. The assay is described under “Experimental Procedures” and uses purified recombinant proteins as in Fig. 2A. Data are derived from three independent experiments. The Kd is 3.5 M. Error bars are the standard deviation from the mean of three replicates.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: In Vitro, Binding Assay, Purification, Recombinant, Control, Incubation, Immunoprecipitation, Western Blot, Competitive ELISA, Derivative Assay, Standard Deviation

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 3. Co-precipitation of AF9 and Hsp90 in vivo. A, HeLa cells were transfectedwithCMV-FLAG-AF9vectorexpressingfull-lengthAF9orwiththe empty vector (CMV-FLAG) as a control. Cell lysates were immunoprecipitated with an antibody that recognizes both Hsp90 isoforms (or IgG control). The precipitated complexes were analyzed by Western blot for FLAG to detect FLAG-tagged AF9. The figure is representative of three experiments. B, non- transfected whole cell lysates were prepared from IMCD3 cells and immuno- precipitatedasinFig.3A.EndogenousAF9wasdetectedbyWesternblotwith an AF9 antibody. The figure is representative of three experiments. C, anti- bodies specific for the and isoforms of Hsp90 were used to immunopre- cipitate proteins from IMCD3 cells. IgG served as a negative control. Endoge- nous AF9 was detected by Western blot. The figure is representative of three experiments.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: In Vivo, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Transfection, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 5. Effects of pharmacologic inhibition and siRNA knock-down of Hsp90 on AF9. A, IMCD3 cells were treated with Hsp90 inhibitors novobiocin (Novo) and 17AAG as well as siRNA directed against both isoforms of mouse Hsp90. Scrambled siRNA served as a control. Whole cell lysates were prepared, and the relative abundance of proteins was assesses by Western blot. siRNA reduces Hsp90 protein abundance. AF9 protein abundance is not affected by any of the interventions. The vertical lines in the Hsp90 Western blot indicate that the position of the lanes was digitally manipulated. Experiment was repeated in triplicate with similar results. B, IMCD3 cells were treated as in Fig. 5A. Cells were then examined by immunofluorescence microscopy with the antibodies and stains indicated in the figure. AF9 shifts from a primarily nuclear localiza- tion to a nuclear and cytoplasmic distribution in cells treated with Hsp90 siRNA or novobiocin. The experiment wasrepeatedintriplicate,anddatafromonesetofexperimentsareshown.Additionalimagescanbefurnished upon request.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: Inhibition, Knockdown, Control, Western Blot, Quantitative Proteomics, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 4. Co-precipitation of AF9 and Hsp70 and the co-chaperone p60/ Hop. IMCD3 cell lysates were immunoprecipitated with an Hsp70 antibody (upper panel) or p60/Hop antibody (lower panel). AF9 was detected in the immune complexesbyWesternblot.Thefigureisrepresentativeofthreeexperiments.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Hsp90 Directly Modulates the Spatial Distribution of AF9/MLLT3 and Affects Target Gene Expression

doi: 10.1074/jbc.m110.101642

Figure Lengend Snippet: FIGURE 7. Analysis of the ENaC promoter region by ChIP. ChIP was performed with Hsp90 and AF9 anti- bodiesandprecipitatedDNAwasamplifiedbyprimersspanningtheregion1372to494relativetothestart site. The primers are described in the text and are derived from the work of Zhang et al. (6) and personal communication with Dr. Zhang. The experiment was repeated in triplicate and fold difference relative to controlwascalculatedusingdensitometryfromtheNIHprogramImageJ.Thecombineddataarepresentedas 95%confidenceintervals(CI)ofatdistributionofunknownvariance.CIsthatfalloutsidethecontrolvalueof1.0 are indicated with a star.

Article Snippet: Immunoprecipitation and Western Blotting—Antibodies against Hsp90 / , Hsp90 , Hsp90 , Hsp70, p60/HOP (all Stressgen), AF9 (Novus), FLAG (Sigma), -actin (Sigma), and IgGcontrol (SantaCruzBiotechnology)were employed.Adilution of 1:1000–1:2500 was used for immunoprecipitation.

Techniques: ChIP-chip, Derivative Assay