anti saa1 2 antibody  (R&D Systems)

Journal: Molecular Metabolism

Article Title: Liver matrin-3 protects mice against hepatic steatosis and stress response via constitutive androstane receptor

doi: 10.1016/j.molmet.2024.101977

Figure Lengend Snippet: The restoration of CAR signaling partially attenuates hepatic steatosis and stress response in the liver of female Matr3 LKO mice. (A) Schematic diagram of experimental timeline for AAV-TBG-GFP or AAV-TBG-GFP-CAR injection and 60 kcal% HFD in female Matr3 fl/fl and Matr3 LKO mice. (B) Real-time qPCR analysis of CAR mRNA expression in the livers of mice. (C) Immunostaining of GFP or GFP-CAR in the livers of mice. (D) Real-time qPCR analysis of Ces2a and Il1r1 mRNA expression in the livers of Matr3 fl/fl and Matr3 LKO mice. (E) The levels of hepatic triglyceride. (F) Oil Red O staining of liver sections and quantification. (G) Real-time qPCR analysis of genes involved in the acute-phase response and immunostaining of SAA1/SAA2 in the livers of Matr3 fl/fl and Matr3 LKO mice. Nuclei are in cyan and SAA1/SAA2 are in magenta. Data are presented as mean ± SEM, n = 7–16 per group; ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Anti-SAA1/2 (Abcam, ab199030) were used at 1:100 dilution.

Techniques: Injection, Expressing, Immunostaining, Staining

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a Summary model depicting the occurrence of lung metastasis at the citrullination site in cancer patients (upper). Replacement of Saa cluster genes in the mouse genome by a human counterpart (middle). Screening of genes related to the pre-metastatic phase using Fbg-deposited pulmonary vessels from noncancer and cancer patients. Generation of murine SAA gene cluster deficient and human SAA gene cluster transgene mice. Genomic structure of mouse and human SAA cluster genes (bottom). b Summary of microarray data. Venn diagram of genes with an over 4-fold increase in mRNA expressions comparing No. 1 vs. No. 4, No. 2 vs. No. 4, and No. 3 vs. No. 4 depicted in Fig. 1a. Gene names and accession numbers for No. 2 vs. No. 4 are shown in supplementary Table . c SAA3 and SAA1/2 proteins were colocalized with Fbg in LLC-bearing mouse lungs. Scale bar, 50 μm. d Representative immunohistochemistry (IHC) photo of SAA3 expression in endothelial cells (EC) of tumor-conditioned media (TCM)-stimulated mouse lungs. Scale bar, 50 μm. e SAA3 induction in LLC-bearing-wild-type but not -SAAs−/− mice (left) ( n = 3 WT-no tumor; n = 6 WT-tumor; n = 4 SAAs−/−-no tumor; n = 6 SAAs−/−-tumor), and Fbg induction (right) ( n = 6 WT-no tumor; n = 8 WT-tumor; n = 4 SAAs−/−-no tumor; n = 6 SAAs−/−-tumor). One-way ANOVA with Bonferroni correction. Mean ± SEM. f Appearance of a shifted band of Fbg in the total lysate and SAA3-IP samples prepared from primary tumor-stimulating lungs. Arrow shows a citrullinated α chain of Fbg. Two independent experiments. g Fbg bound to SAA3 in LLC-bearing lungs was assumed to be citrullinated (arrow). Immunoprecipitated SAA3 (left bottom) was also presented in the western blot. Two independent experiments. h The citrullinated Fbg band in tumor-bearings in a SAAs-dependent manner (arrow). Two anti-citrullination antibodies were used. Three independent experiments. i , j Immunohistochemical colocalization signal between citrulline and Fbg on MECA32 + -endothelial cells in LLC-bearing mouse lungs ( i ) and the signal was prominent in wild-type compared to SAAs−/− mice (arrowhead). Scale bar, 20 μm. k Immunohistochemical quantification of citrulline (left) ( n = 8 WT-no tumor; n = 8 WT-LLC tumor; n = 6 SAAs−/−−no tumor; n = 8 SAAs−/−−LLC tumor), Fbg (middle), and citrullinated Fbg (right) ( n = 4 WT-no tumor; n = 5 WT-LLC tumor; n = 4 SAAs−/−−no tumor; n = 4 SAAs−/−−LLC tumor) in no tumor-bearing- and LLC-bearing-wild-type and SAAs-/- mouse lungs. One-way ANOVA with Bonferroni correction. Mean ± SEM.

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Microarray, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Immunohistochemical staining

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a Quantifications of PAD4 in endothelial cells (EC) (PAD4-EC) ( n = 5 WT-no tumor; n = 6 WT-LLC tumor; n = 5 SAAs-/--no tumor; n = 6 SAAs-/--LLC tumor), CD11b + cells (PAD4-CD11b) ( n = 5 WT-no tumor; n = 5 WT-LLC tumor; n = 5 SAAs-/--no tumor; n = 5 SAAs-/--LLC tumor) and F4/80 + cells (PAD4-F4/80) ( n = 5 WT-no tumor; n = 5 WT-LLC tumor; n = 5 SAAs-/--no tumor; n = 5 SAAs-/--LLC tumor) in no tumor-bearing and LLC-bearing lung tissues in IHC analysis. One-way ANOVA with Bonferroni correction. Mean ± SEM. b Cell-mediated citrullination assay scheme. The same number of CD144 + EC, CD11b + , and F4/80 + cells derived from tumor-bearing lungs were applied on various concentrations of Fbg-coated plates. Fbg citrullination was evaluated by IP-western blot (Fig. 2c). c Citrullination ability in purified EC compared with CD11b + cells and F4/80 + cells. Three independent experiments. d Measurement of citrullination area of Fbg-coated plate by purified cells from tumor-bearing-wild-type and SAAs−/− mouse lungs. Mean ± SEM. Three independent experiments. e Citrullination after knockdown of PAD2 and PAD4 in ECs and CD11b + cells derived from tumor-bearing mouse lungs. Immunohistochemical image of ECs (upper) and quantification of PAD and citrullination signals normalized by DAPI (ECs). To calculate the PADs signals of CD11b + cells, DAPI normalization was omitted because extracellular PADs from these cells should be considered (lower). Mean ± SEM (Student’s two-sided t -test). Scale bar, 50 μm. Three independent experiments. f Pre-metastatic and post-metastatic models (upper). Mouse-holding tumors that did not spontaneously metastasize were defined as pre-metastatic. A pre-metastatic mouse that received an injection of metastatic tumor cells in the blood vessel was defined as post-metastatic. The short distance between post-metastatic cells and SAAs-CitFbg implies a presumptive pre-metastatic niche (lower). g Number of metastatic cells in lungs after the tumor cell injection to LLC-bearing and E0771-bearing mice. For LLC-bearing mouse and E0771-bearing mouse analyses, 60 and 56 sections of WT and SAAs−/− lungs were examined, respectively. In the analysis, two to three sections per lobe were chosen, and all lobes (from five WT and five SAAs−/− mice) were examined. Mean ± SEM (Student’s two-sided t -test). h Recruitment of tumor cells around SAA1/2-Fbg, SAA3-Fbg, and CitFbg in LLC-bearing mice. Tumor cells can be identified by the blue pseudo-color in merged SAA signals. Scale bars, 20 μm. Six independent experiments. i Mirror section to detect tumor cells near the SAA3-CitFbg signals in the lung. IHC analysis for two consecutive frozen sections, No. 1, and No. 2, with the cut surfaces facing each other upwards for staining with different antibodies. Scale bars, 20 μm. Two independent experiments. j Kinetic distance between tumor cells and proteins. Data analysis based on a staining set of SAA3/Fbg (left), citrulline/Fbg (middle), and CitFbg/Fbg (right) in tumor-bearing wild-type and SAAs−/− mice (areas were chosen from n = 5 wild-type mice and from n = 5 SAAs−/− mice, respectively). Mean ± SEM. Two-way ANOVA.

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Derivative Assay, Western Blot, Purification, Immunohistochemical staining, Injection, Staining

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a IHC analysis of CitFbg in the alveolar region of noncancer and cancer patients with a specific antibody for hCitFbg that was validated to have no cross-reaction for fibrin. Lung tissue samples with no microscopical metastases were used. b The DAB signals in capillaries of cancer patients were shown. Scale bars, 200 μm. Four independent experiments. c Representative images with double staining of CitFbg/VE-cadherin and CitFbg/SAA1/2 and CitFbg/Fbg. Arrows indicate the same vessels. Scale bars, 10 μm. d – h Quantification of the area of CitFbg ( d ), Fbg ( e ), CitFbg/Fbg ( f ), SAA1/2 ( g ), and CitFbg/SAA1/2 ( h ) signals in small pulmonary vessels ( n = 16 no cancer, n = 29 cancer). Data were mean ± SEM (Student’s two-sided t -test). i Ratio of the area of CitFbg in Fbg ( n = 16 no cancer; n = 29 cancer). Data were mean ± SEM (Student’s two-sided t -test). j IHC analysis using various regions with no evidence of tumor cells derived from no-metastatic lobes, no-metastatic, or metastatic regions in lobes with metastasis in the same cancer patients. k Representative images of CitFbg/VE-cadherin, CitFbg/SAA1/2, and CitFbg/Fbg. Arrows indicate the same position. Scale bars, 200 μm. l – p Quantification of the area of CitFbg ( l ), Fbg ( m ), CitFbg/Fbg ( n ), SAA1/2 ( o ), and CitFbg/SAA1/2 ( p ). q Ratio of the area of CitFbg in Fbg ( n = 6 no metastasis; n = 7 metastasis). One-way ANOVA with Bonferroni correction. Data were mean ± SEM.

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Double Staining, Derivative Assay

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a Scheme of hSAAs-hCitFbg complex-induced metastasis. First, PBS, hFbg, or hCitFbg was injected into humanized hSAAs-Tg/mSAAs-KO mice. Then, labeled human cancer cells were injected. b Detection of SAAs proteins in lung tissues after injecting hCitFbg into hSAAs/mSAAs-/- mice by western blot analysis. The mSAAs-/-mice were used as a negative control. Two independent experiments. c Numbers of metastatic MCF7 ( n = 4 hSAAs/mSAAs-/-; n = 3 mSAAs-/-) and MDAMB231 cells ( n = 4 hSAAs/mSAAs-/--PBS; n = 7 hSAAs/mSAAs-/--hFbg and -hCitFbg; n = 7 SAAs-/-) in PBS-, hFbg-, and hCitFbg- treated-hSAAs/mSAAs-/- and mSAAs-/-mice. One-way ANOVA with Bonferroni correction. Data were mean ± SEM. d Representative IHC photos of metastatic MCF7 cells trapped by the hSAAs-hCitFbg complex. Scale bars, 20 μm. e Distances among hCitFbg, hSAA1/2, hSAA4, and MCF7 cells shown in ( d ) were analyzed ( n = 4 hSAAs/mSAAs-/-; n = 3 mSAAs-/-) One-way ANOVA with Bonferroni correction. Data were mean ± SEM. f Summary of the distance among metastatic tumor cells, SAA3-hCitFbg, and hSAA1/2-hCitFbg. ( n = 5 LLC-C57BL/6; n = 5 E0771-C57BL/6; n = 4 LLC-Scid; n = 3; MCF7- hSAAs/mSAAs-/-; n = 4 MDAMB231- hSAAs/mSAAs-/-) Data were mean ± SEM (Student’s two-sided t -test). g Similar strategy to Fig. 5a in hSAAs-Tg/mSAAs-KO in Rag1-/- background (upper left). Representative staining of Ki67 in lung 3 weeks after injection of tumor cells in hSAAs/mSAAs-/-/Rag1-/- and mSAAs-/-/Rag1-/- mice (upper right). Quantification of metastatic surface area in lungs 3 weeks after application of MCF7 ( n = 5 mSAA-/-hSAAs-Tg/-Rag1-/-; n = 4 mSAA-/-hSAAs-/-Rag1-/-) and MDAMB231 ( n = 4 mSAA-/-hSAAs-Tg/-Rag1-/-; n = 4 mSAA-/-hSAAs-/-Rag1-/-) cells (lower). Data were mean ± SEM (Student’s two-sided t -test).

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Injection, Labeling, Western Blot, Negative Control, Staining

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a Enzyme-linked immunosorbent assay (ELISA) for the binding between hSAA1 and hFbg or between hSAA1 and hCitFbg. Data points show mean ± SEM of four independent experiments. b Gel filtration analysis of hFbg and hCitFbg in the absence and presence of hSAA1 (left). The eluted fraction (fraction 4) corresponding to hSAA1-hFbg and hSAA1-hCitFbg was analyzed using a western blot (right). The intensities of the bands corresponding to hFbg, hCitFbg, and hSAA1 were quantified (lower). Three independent experiments. c Dynamic light scattering (DLS) analysis of Fbg and CitFbg in the presence and absence of SAA1 (left). The averaged hydrodynamic diameters were listed (right). d Formation of aggregations in CM after application of Fbg, CitFbg, and SAA1 in the presence of human lung micro-vessel ECs (lower left). Representative images of protein aggregations (upper). Large-size aggregations were counted in comparison with those of naturally secreted SAA1 from ECs (lower right). One-way ANOVA with Bonferroni correction. Data were mean ± SEM. Scale bars, 25 μm. Four independent experiments. e Degradation kinetics of Fbg, CitFbg, SAA1-Fbg, and SAA1-CitFbg with plasmin were shown in PAGE-Coomassie blue staining (upper left) and Western blot analysis (upper right, reducing condition). Comparison of degradation rates of Fbg vs. CitFbg (Data points show mean ± SEM of two independent experiments) and SAA1-Fbg vs. SAA1-CitFbg (lower).

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Filtration, Western Blot, Staining

Journal: Nature Communications

Article Title: Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis

doi: 10.1038/s41467-023-40371-1

Figure Lengend Snippet: a , b Human tumor cell aggregation assay for measuring Fbg, CitFbg, SAA1-Fbg, and SAA1-CitFbg proteins. Representative photo of aggregated MDAMB231 cells with SAA1-CitFbg protein complex (Scale bars, 50 μm in ( a ). The cell aggregation number (five or more cells in aggregation) of MCF7 and MDAMB231 cells cultured on various protein complex-coating plates for 3 days ( b ). ( n = 3 for MCF7 and n = 4 for MDAMB231 cells). One-way ANOVA with Bonferroni correction. Data were mean ± SEM. c Screening of CitFbg peptides in inhibition assay against the tumor cell attachment to Fbg and CitFbg ( n = 3). FGA-1, FGA-C(citrulline)-2, FGA-3, and FGA-C-4 were 15 amino acid length peptides derived from the N-terminal or C-terminal of the Fbg and CitFbg protein sequences, respectively. One-way ANOVA with Bonferroni correction. Data were mean ± SEM. d Competitive inhibition assay in vitro against the tumor cell attachment to Fbg and CitFbg using N-terminal peptides of Fbg and CitFbg. MCF7 cells ( n = 10) and MDAMB231 cells ( n = 5) were shown after applying the appropriate peptides. Data were mean ± SEM. Two independent experiments. e In vivo metastasis inhibition assay using FGA-1 and FGA-C−2 peptides. The number of metastatic tumor cells in the lung after application of the CitFbg protein, followed by the peptides. ( n = 3 C.B17/Icr-scidJcl for MCF7 and n = 5 C.B17/Icr-scidJcl for MDAMB231). One-way ANOVA with Bonferroni correction. Data were mean ± SEM. f Model to detect and suppress pre-/early-metastatic sites in cancer patients. A retrospective study of cancer patients indicated that hSAAs-hCitFbg is a presumptive pre-metastatic molecule. A prospective study demonstrated that the formation of hSAAs-hCitFbg recruits cancer cells to facilitate metastasis.

Article Snippet: For human transgenic SAA staining, we used three antibodies for human SAA1/2: (1) ab200584 (1:100, Abcam), (2) ab207445 (1:100, Abcam), and (3) ab687 (1:100, Abcam).

Techniques: Cell Culture, Inhibition, Cell Attachment Assay, Derivative Assay, In Vitro, In Vivo