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: Cell reports

Article Title: Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer

doi: 10.1016/j.celrep.2022.111596

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-SAA1 , Biorbyt , Cat #orb228668; RRID:AB_2924684.

Techniques: Plasmid Preparation, Polymer, Staining, Virus, Recombinant, Sequencing, Software

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Alignment of amino acid sequences of mouse serum amyloid A (SAA) isoforms. The sequences (AAEKISDGREAFOE and QRWVQFMKEAG) of synthetic SAA1 and SAA3 peptides for immunization to rabbits are underlined. Accession numbers are shown in parentheses.

Article Snippet: Subsequently, the membrane was incubated with the primary anti-mouse SAA1 rabbit polyclonal antibody (1:500, #PAA885Mu01, Cloud-Clone, Houston, TX, USA) or anti-SAA3 rat monoclonal antibody [JOR110A] (1:50, #ab231680, Abcam, Cambridge, UK) diluted with 1% nonfat dried skim milk in TBST for 1 h at room temperature.

Techniques:

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Oligonucleotide primers used in quantitative real-time PCR.

Article Snippet: Subsequently, the membrane was incubated with the primary anti-mouse SAA1 rabbit polyclonal antibody (1:500, #PAA885Mu01, Cloud-Clone, Houston, TX, USA) or anti-SAA3 rat monoclonal antibody [JOR110A] (1:50, #ab231680, Abcam, Cambridge, UK) diluted with 1% nonfat dried skim milk in TBST for 1 h at room temperature.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Comparison of Saa1 and Saa3 mRNA expression levels in NMuMG cells treated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Data are presented as the mean ± standard deviation of four independent experiments. Asterisks indicate significant difference compared with control levels: ** p < 0.01. NMuMG, normal murine mammary gland.

Article Snippet: Subsequently, the membrane was incubated with the primary anti-mouse SAA1 rabbit polyclonal antibody (1:500, #PAA885Mu01, Cloud-Clone, Houston, TX, USA) or anti-SAA3 rat monoclonal antibody [JOR110A] (1:50, #ab231680, Abcam, Cambridge, UK) diluted with 1% nonfat dried skim milk in TBST for 1 h at room temperature.

Techniques: Expressing, Standard Deviation

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Comparison of SAA1 (A and B) and SAA3 (C and D) protein expression levels in NMuMG cells treated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by immunofluorescence analysis (IFA). ( A , C ) Fields of view where fluorescence intensity was measured. Scale bar = 20 µm. ( B , D ) The relative SAA1 and SAA3 protein expression levels in NMuMG cells following stimulation with LPS or LTA were normalized to those in untreated, control cells. Data are presented as the mean fluorescence of five or more random locations with vertical bars representing standard deviation. Asterisks indicate significant difference compared with control levels: * p < 0.05, ** p < 0.01. NMuMG, normal murine mammary gland.

Article Snippet: Subsequently, the membrane was incubated with the primary anti-mouse SAA1 rabbit polyclonal antibody (1:500, #PAA885Mu01, Cloud-Clone, Houston, TX, USA) or anti-SAA3 rat monoclonal antibody [JOR110A] (1:50, #ab231680, Abcam, Cambridge, UK) diluted with 1% nonfat dried skim milk in TBST for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Fluorescence, Standard Deviation

Journal: Animals : an Open Access Journal from MDPI

Article Title: Induction of Serum Amyloid A3 in Mouse Mammary Epithelial Cells Stimulated with Lipopolysaccharide and Lipoteichoic Acid

doi: 10.3390/ani11061548

Figure Lengend Snippet: Detection of SAA1 and SAA3 protein expression in NMuMG cell supernatants treated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA) by Western blot. ( A ) Detection of SAA1 in the cell supernatant. Membranes were incubated with the primary anti-mouse SAA1 antibody (dilution of 1:500, 60 s exposure time). ( B ) Detection of SAA3 in the cell supernatant. Membranes were incubated with the primary anti-mouse SAA3 antibody (dilution of 1:50, 120 s exposure time). PC, positive control; 1, recombinant murine SAA1 (rSAA1); 3, rSAA3; NMuMG, normal murine mammary gland.

Article Snippet: Subsequently, the membrane was incubated with the primary anti-mouse SAA1 rabbit polyclonal antibody (1:500, #PAA885Mu01, Cloud-Clone, Houston, TX, USA) or anti-SAA3 rat monoclonal antibody [JOR110A] (1:50, #ab231680, Abcam, Cambridge, UK) diluted with 1% nonfat dried skim milk in TBST for 1 h at room temperature.

Techniques: Expressing, Western Blot, Incubation, Positive Control, Recombinant

Journal: Oncogene

Article Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4.

doi: 10.1038/onc.2013.568

Figure Lengend Snippet: Figure 1. S100A4 specifically upregulates SAA in VMR cells. SAA1 (A) and SAA3 (B) expression in VMR cells incubated with recS100A4 ( þ S100A4) for the indicated times was analyzed by qPCR, and normalized relative to non-treated controls (fold induction). Insets: Western blots of SAA1 and SAA3 in CM from VMR cells treated with ( þ ) and without ( ) recS100A4 for 24 h. The experiments were performed independently three times, each with three replicates for each time point. Representative western blot analyses are presented in the insets. (C) Western blot of SAA1 in CM from the indicated human cell lines treated with ( þ ) and without ( ) recS100A4 for 24 h. Loading control: actin. (D) SAA1 and SAA3 protein accumulation in CM from VMR cells incubated with the indicated recS100 proteins in the presence of PMB. (E) (a–c) Western blot of SAA in CM from VMR cells after stimulation with human S100A4 (hA4) in the presence of two different anti-human S100A4 monoclonal antibodies (Ab no. 1 and Ab no. 2) that inhibit SAA1 and SAA3 upregulation in response to hA4 but not mouse S100A4 (moA4). Non-treated cells (CTL) and isotype control antibodies (IgG) served as controls. Tubulin served as a loading control. (D) Mutated forms of moA4 (moA4mt1 and moA4mt2) unable to form oligomers and have an attenuated ability to induce SAA3. Representative western blot analysis is presented.

Article Snippet: Other antibodies used were rabbit anti-mouse SAA3 serum,30 rabbit anti-mouse SAA1 antibodies (a kind gift from Lars Bo Nielsen, Copenhagen University, København K, Denmark),48 mouse monoclonal anti-actin antibody AC-40 (Sigma-Aldrich), rabbit anti-MMP-3 antibodies (Epitomics, Burlingame, CA, USA) and rabbit anti-NF-kB p65 antibodies (Santa Cruz, Heidelberg, Germany).

Techniques: Expressing, Incubation, Western Blot, Control, Bioprocessing

Journal: Oncogene

Article Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4.

doi: 10.1038/onc.2013.568

Figure Lengend Snippet: Figure 4. S100A4 and SAA signal via TLR4 and NF-kB. (a) VMR cells were treated with S100A4 (A4) or lipopolysaccharide in the presence and absence of TLR4, IRAK and NF-kB inhibitors. Expression of SAA1 in CM from the cells was assessed by western blotting. Representative western blot analysis from two independent experiments is presented. (b) VMR-I-kB and control cells (VMR-CTL) were engineered to express a NF-kB- driven luciferase reporter, and then were treated with S100A4 and tumor necrosis factor-a. Luciferase activity relative to mock-treated cells (CTL) was assessed. The experiments were performed independently three times, each with three replicate samples. (c) VMR cells were treated with S100A4 in the presence or absence of EGFR or MEK inhibitors. Expression of SAA3 relative to mock-treated cells was assessed by qPCR, and by western blot (insert). The experiments were performed independently four times, each with three replicate samples. A representative western blot is presented. (d) Western blots of SAA1 in CM from VMR cells treated with recS100A4, recSAA1 or recSAA3 in the presence and absence of PMB or inhibitors of TLR4 and NF-kB. Both the added recSAA1 and the endogenous cell-derived SAA1 are visible. A representative western blot analysis from two independent experiments is presented.

Article Snippet: Other antibodies used were rabbit anti-mouse SAA3 serum,30 rabbit anti-mouse SAA1 antibodies (a kind gift from Lars Bo Nielsen, Copenhagen University, København K, Denmark),48 mouse monoclonal anti-actin antibody AC-40 (Sigma-Aldrich), rabbit anti-MMP-3 antibodies (Epitomics, Burlingame, CA, USA) and rabbit anti-NF-kB p65 antibodies (Santa Cruz, Heidelberg, Germany).

Techniques: Expressing, Western Blot, Control, Luciferase, Activity Assay, Derivative Assay

Journal: Oncogene

Article Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4.

doi: 10.1038/onc.2013.568

Figure Lengend Snippet: Figure 5. Ectopic SAA expression stimulates the metastatic potential of VMR tumor cells. VMR-SAA3 or VMR-CTL were used in spontaneous (a and b) and experimental (c and d) metastasis assays (nine mice per group). (e and f) VMR-SAA1 was used in experimental metastasis assays (eight mice per group). Numbers of metastases in liver and lungs were evaluated. (g) Immunohistochemical staining with CD45 antibodies of sections of lung (upper panel) and liver (lower panel) from VMR-CTL and VMR-SAA3 tumor-bearing mice. Scale bars, 50 mm. Immunohistochemical stainings were repeated two times and representative data are presented.

Article Snippet: Other antibodies used were rabbit anti-mouse SAA3 serum,30 rabbit anti-mouse SAA1 antibodies (a kind gift from Lars Bo Nielsen, Copenhagen University, København K, Denmark),48 mouse monoclonal anti-actin antibody AC-40 (Sigma-Aldrich), rabbit anti-MMP-3 antibodies (Epitomics, Burlingame, CA, USA) and rabbit anti-NF-kB p65 antibodies (Santa Cruz, Heidelberg, Germany).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncogene

Article Title: A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4.

doi: 10.1038/onc.2013.568

Figure Lengend Snippet: Figure 6. S100 proteins induce SAA and cytokine expression in an organ-specific manner. RecS100A4, recS100A8 and recS100A9 was injected intravenously into mice for 2 weeks. Expression of SAA1 (a and e), SAA3 (b and f), RANTES and G-CSF (c and g), and S100A8 and S100A9 (d and h) in lungs, liver and spleen was then assessed using qPCR, relative to the mean of the PBS control (fold induction). (i) SAA3 protein was detected in western blots of serum prepared from animals treated with recS100A4, recS100A8 and recS100A9 proteins. Seven or eight mice were used in each group.

Article Snippet: Other antibodies used were rabbit anti-mouse SAA3 serum,30 rabbit anti-mouse SAA1 antibodies (a kind gift from Lars Bo Nielsen, Copenhagen University, København K, Denmark),48 mouse monoclonal anti-actin antibody AC-40 (Sigma-Aldrich), rabbit anti-MMP-3 antibodies (Epitomics, Burlingame, CA, USA) and rabbit anti-NF-kB p65 antibodies (Santa Cruz, Heidelberg, Germany).

Techniques: Expressing, Injection, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased IL-17-expressing γδT cells in SAA1 TG mice. A , left , flow cytometry detection of lymph node IL-17A + γδ T cell receptor (TCR) + cells, ( B , left ) IL-17A + CD4 + (Th17) cells, and ( C , left ) lineage − CD45 + CD90.2 + IL-17A + (ILC3) cells, represented by dot blots. Data are representative of quadruplicate experiments. ( A – C , right , upper ) The percentages of IL-17A + γδ TCR + , Th17, and ILC3 cells in 5000 lymph node cells (P1) are shown (n = 4). ( A – C , right , bottom ) The absolute numbers of IL-17A + γδ TCR + , Th17, and ILC3 cells per 5000 lymph node cells (P1) are shown (n = 4). Detailed gating strategies and information for P1 are shown ( Fig. S1 ). Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased IL-17-expressing neutrophils in SAA1 TG mice. A , upper , circulating G-CSF levels measured by western blot using equal volumes (1 μl) of serum. The molecular weight of G-CSF is 19 kDa ( A , bottom ). The ponceau S staining to show that the same volume of serum was loaded is shown ( Fig. S2 ). Densitometry analysis of western blots showing G-CSF quantities, n = 3. B , left , blood CD11b + Ly6G + cells (neutrophils) detected by flow cytometry are represented by a dot blot, n = 4. B , right , the percentages of CD11b + Ly6G + cells and absolute numbers of CD11b + Ly6G + cells per 5000 blood cells (P1) are shown (n = 4). C , left , CD11b + Ly6G + cells gated to IL-17A + Ly6G + cells (IL-17-producing neutrophils) by flow cytometry are represented by a dot blot (n = 4). C , right , the percentage of IL-17A + CD11b + Ly6G + cells and absolute numbers of cells per 5000 blood cells (P1) are shown (n = 4). Detailed gating strategies and information for P1 are shown ( Fig. S3 ). Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 compared with WT mice. The red arrow indicates that A is gated to B . MW, molecular weight.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Molecular Weight, Staining, Flow Cytometry, Dot Blot, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased osteoclast-related gene expression and IL-17 and IL-23 expression in whole bone lysates from 6-month-old SAA1-overexpressing TG mice. A , upper , circulating SAA1 levels measured by western blot using equal volumes (1 μl) of serum from 6-month-old mice. The molecular weight of SAA1 is 12 kDa ( A , bottom ). Densitometry analysis of western blots showing SAA1 quantities, n = 3. The ponceau S staining to show that the same volume of serum was loaded is shown ( Fig. S2 ). Relative mRNA expression levels of ( B ) Ctsk , Tnfsf11 , Nfatc1 , and Acp5 ( C ) Tnfrsf11b , Alpl , Bglap , and Runx2 ( D ) Axin1 , Ctnnb1 , and Ccn4 , and ( E ) Il17a and Il23a , normalized to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA expression levels, and relative to WT mice in whole bone lysates. For B–E ; WT; n = 4 and TG; n = 8. Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice. MW; molecular weight.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Molecular Weight, Staining, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased Tnfsf11 mRNA expression levels in bone marrow from 6-month-old SAA1-overexpressing TG mice. A , relative mRNA expression levels of Tnfsf11 normalized to glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) mRNA expression levels and relative to WT mice in bone lysates. B , Tnfsf11/Gapdh ΔCT was calculated by subtracting the CT value for mRNA Gapdh expression levels from the CT value for Tnfsf11 mRNA expression levels. Data are expressed as the mean ± standard deviation, and unpaired two-tailed Student's t -test was applied to compare two groups. For A and B ; WT; n = 4, TG; n = 5. N.D; not detected.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased populations of RANKL-expressing cells in bone marrow from SAA1-overexpressing TG mice. A , left , Total RANKL + cells and ( B , left ) CD45 + RANKL + and CD45 − RANKL + cells in bone marrow were detected by flow cytometry and represented by a dot blot (n = 4). Representative data from quadruplicate experiments are shown. The percentage and absolute numbers of ( A , right ) RANKL + cells and ( B , middle ) CD45 + RANKL + and CD45 − RANKL + cells per 5000 of bone marrow cells (P1) are shown (n = 4). B , right , detailed gating strategies and information for P1 are shown ( Fig. S4 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Dot Blot, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased RANKL-expressing innate immune cells in the bone marrow from 6-month-old SAA1 TG mice. A , left , γδ TCR + cells and ( B , left ) CD11b + Ly6G + cells in bone marrow were detected by flow cytometry and represented by dot blots (n = 4). Representative data from quadruplicate experiments are shown. The percentages and absolute numbers of ( A , right ) γδ TCR + cells and ( B , right ) CD11b + Ly6G + cells per 10,000 bone marrow cells (P1) are shown. Detailed gating strategies and information for P1 are shown ( Fig. S5 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Increased RANKL-expressing neutrophils in bone marrow from SAA1 TG mice. A , left , RANKL + γδTCR + cells and ( B , left ) RANKL + CD11b + Ly6G + cells in bone marrow were detected by flow cytometry and represented by dot blots (n = 4). Representative data from quadruplicate experiments are shown. A , right , upper , the percentage of RANKL + γδTCR + cells and ( A , right , bottom ), the counts for RANKL + γδTCR + cells in 10,000 bone marrow cells (P1) are shown (n = 4). B , right , upper , the percentage of RANKL + cells and ( B , right , bottom ), the counts for RANKL + cells per 5000 CD11b + Ly6G + cells in bone marrow (E1) are shown (n = 4). Detailed gating strategies and information for P1 and E1 are shown ( Fig. S6 ). Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Standard Deviation, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Overexpression of hepatic serum amyloid A1 in mice increases IL-17-producing innate immune cells and decreases bone density

doi: 10.1016/j.jbc.2021.100595

Figure Lengend Snippet: Changes in bone mineral density and architecture in SAA1 TG mice. A , upper , bone mineral density, bone volume/total volume, trabecular thickness, trabecular number, and cortical thickness of SAA1 TG and WT mice (n = 8). B , a representative TRAP stained section from a quadruplicate experiment is shown. The red arrows indicate TRAP-positive areas (n = 4). (WT, TG, left ) scale bar = 500 μm, (WT, TG, middle , right ) scale bar = 100 μm. Data are expressed as the mean ± standard deviation and unpaired two-tailed Student's t -test was applied to compare two groups. ∗ p < 0.05 and ∗∗ p < 0.01 compared with WT mice.

Article Snippet: Goat anti-mouse SAA1 antibody (Abcam) diluted at a ratio of 1:1000, Goat anti-G-CSF primary antibodies (Santa Cruz Biotechnology) diluted at a ratio of 1:2000 were used, followed by HRP-conjugated anti-goat IgG secondary antibodies (Santa Cruz Biotechnology).

Techniques: Staining, Standard Deviation, Two Tailed Test