Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: Increased MDSCs and SAA1 expression associated with ovarian cancer. ( A ) Flow cytometry analysis of M-MDSCs in PBMCs from patients with benign and malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( B ) Flow cytometry analysis of PMN-MDSCs in PBMCs from patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( C ) Flow cytometry analysis of M-MDSCs in tumor-infiltrating immune cells from patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( D ) Flow cytometry analysis of PMN-MDSCs in tumor-infiltrating immune cells from patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( E ) Flow cytometry analysis of Treg cells in PBMCs from patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( F ) qRT-PCR analysis of the mRNA expression levels of iNOS, IDO and Arg-1 in tumor tissues from patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( G ) Heatmap showing the top upregulated differentially expressed genes following co-culture of SKOV3 cells and MDSCs based on analysis of the GSE145374 dataset. ( H ) SAA1 expression in ovarian cancer tissues based on the GEPIA database. Each dot represents one sample; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( I ) Kaplan–Meier analysis of overall survival in ovarian cancer patients stratified by SAA1 expression using GEPIA. Log-rank test. ( J ) Detection of SAA1 mRNA expression in benign and malignant ovarian tumor tissues by qRT-PCR. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( K ) Detection of SAA1 protein levels in serum from patients with benign and malignant ovarian tumors by ELISA. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( L ) Association between SAA1 expression and FIGO stage in ovarian cancer based on the public GSE51088 dataset. Each dot represents one sample; Mann–Whitney U test. ( M ) Association between SAA1 expression and histological grade in ovarian cancer based on the public GSE63885 dataset. Each dot represents one sample; Kruskal–Wallis test. ( N ) SAA1 protein expression in paired tumor (T) and adjacent paracancerous (P) tissues assessed by Western blotting (left) and densitometric analysis (right). Each dot represents an individual patient; paired two-tailed Student’s t-test. ( O ) Localization and expression of SAA1 in tumor and paracancerous tissues detected by immunohistochemistry ( n = 3). Representative images are presented at 100× and 400× magnification. ( P ) SAA1 mRNA expression in normal ovarian epithelial cells and ovarian cancer cell lines assessed by qRT-PCR. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( Q ) Detection of SAA1 protein expression in normal ovarian epithelial cells and ovarian cancer cell lines by Western blotting (left) and densitometric analysis (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. *Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: Expressing, Flow Cytometry, MANN-WHITNEY, Quantitative RT-PCR, Co-Culture Assay, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: SAA1 promotes the proliferation of ovarian cancer cells in vitro. ( A ) Detection of SAA1 knockdown efficiency at the mRNA level in A2780 cells by qRT-PCR. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( B–C ) Detection of SAA1 knockdown efficiency at the protein level in A2780 cells by Western blotting ( B ) and densitometric analysis ( C ). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( D ) Detection of SAA1 overexpression efficiency at the mRNA level in SKOV3 cells by qRT-PCR. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( E–F ) Detection of SAA1 overexpression efficiency at the protein level in SKOV3 cells by Western blotting ( E ) and densitometric analysis ( F ). Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( G ) Detection of secreted SAA1 levels in the supernatant of A2780 cells after SAA1 knockdown by ELISA. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( H ) Detection of secreted SAA1 levels in the supernatant of SKOV3 cells after SAA1 overexpression by ELISA. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( I ) CCK8 assay to evaluate the effect of SAA1 knockdown on A2780 cell proliferation. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( J ) CCK8 assay to evaluate the effect of SAA1 overexpression on SKOV3 cell proliferation. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( K–L ) EdU assay to evaluate the proliferative capacity of A2780 cells following SAA1 knockdown. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( M–N ) EdU assay to evaluate the proliferative capacity of SKOV3 cells following SAA1 overexpression. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( O–P ) Colony formation assay to assess the clonogenic capacity of A2780 cells following SAA1 knockdown. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( Q–R ) Colony formation assay to assess the clonogenic capacity of SKOV3 cells following SAA1 overexpression. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. *Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: In Vitro, Knockdown, Quantitative RT-PCR, Western Blot, Over Expression, Two Tailed Test, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, EdU Assay, Colony Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: SAA1 promotes the proliferation and metastasis of ovarian cancer in vivo and in vitro. ( A–C ) Transwell assay to evaluate the migration and invasion capacity of A2780 cells following SAA1 knockdown. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( D–F ) Transwell assay to evaluate the migration and invasion capacity of SKOV3 cells following SAA1 overexpression. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( G–I ) Transwell assay to evaluate the migration and invasion capacity of ID8 cells following SAA1 knockdown. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-test. ( J ) Detection of EMT-related proteins (N-Cadherin, E-Cadherin, Vimentin, Snail) in A2780 cells following SAA1 knockdown by Western blotting (top) and densitometric analysis (bottom). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( K ) Detection of EMT-related proteins in SKOV3 cells following SAA1 overexpression by Western blotting (top) and densitometric analysis (bottom). Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-tests. ( L ) Detection of EMT-related proteins in ID8 cells following SAA1 knockdown by Western blotting (top) and densitometric analysis (bottom). Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-tests. ( M–N ) Luciferase-based in vivo imaging to evaluate the effect of SAA1 knockdown on ovarian cancer progression in tumor-bearing mice. Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( O ) Kaplan–Meier analysis to evaluate the effect of SAA1 knockdown on the survival duration of tumor-bearing mice. Log-rank test. ( P–Q ) Immunohistochemical detection of SAA1, Ki67, E-Cadherin, and N-Cadherin expression in abdominal wall tumors of tumor-bearing mice ( P ) and quantification of positive staining ( Q ). Data are presented as mean ± SEM from three mice per group; unpaired two-tailed Student’s t-tests. *Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: In Vivo, In Vitro, Transwell Assay, Migration, Knockdown, Over Expression, Two Tailed Test, Western Blot, Luciferase, In Vivo Imaging, Immunohistochemical staining, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: SAA1 recruits MDSCs and promotes MDSCs differentiation in ovarian cancer. ( A ) Relationship between SAA1 expression and immune infiltration in ovarian cancer analyzed using the CIBERSORT algorithm based on TCGA data. ( B ) Correlation between SAA1 expression and the infiltration of different immune cell subsets in ovarian cancer using the CIBERSORT algorithm based on TCGA data. ( C–E ) Correlation analysis between SAA1 expression and CD33 ( C ), S100A8 ( D ), or CD14 ( E ) expression in ovarian cancer using the TIMER database; Spearman’s rank correlation test. ( F ) Schematic diagram of MDSCs recruitment and pro-GMP differentiation experiments. ( G–H ) Co-culture assays to detect the ability of supernatants from A2780 cells with SAA1 knockdown to recruit MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( I–J ) Co-culture assays to detect the ability of supernatants from ID8 cells with SAA1 knockdown to recruit MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( K–L ) Co-culture assays to detect the ability of supernatants from SKOV3 cells with SAA1 overexpression to recruit MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( M–N ) Flow cytometry to detect the ability of supernatants from A2780 cells with SAA1 knockdown to promote the differentiation of GMPs to MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( O–P ) Flow cytometry to detect the ability of supernatants from SKOV3 cells with SAA1 overexpression to promote the differentiation of GMPs to MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. *Statistical significance: ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: Expressing, Co-Culture Assay, Knockdown, Over Expression, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: SAA1 released by ovarian cancer cells recruits MDSCs and promotes MDSCs differentiation via TLR2/4. ( A – B ) Flow cytometry analysis of TLR2 expression on the surface of PMN-MDSCs ( A ) and M-MDSCs ( B ) from the peripheral blood of patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( C–D ) Flow cytometry analysis of TLR4 expression on the surface of PMN-MDSCs ( C ) and M-MDSCs ( D ) from the peripheral blood of patients with benign or malignant ovarian tumors. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( E–F ) Correlation analysis between SAA1 expression and TLR2 ( E ) or TLR4 ( F ) expression in ovarian cancer using the TIMER database; Spearman’s rank correlation test. ( G–H ) Rescue experiments assessing the effect of adding recombinant SAA1 protein or the TLR2/4 inhibitor SsnB on the ability of A2780 cell supernatants to recruit MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test. ( I–J ) Rescue experiments assessing the effect of recombinant SAA1 protein or SsnB on the ability of ID8 cell supernatants to recruit MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test. ( K–L ) Rescue experiments assessing the effect of adding recombinant SAA1 protein or SsnB on the ability of A2780 cell supernatants to induce GMP differentiation into MDSCs. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Tukey’s multiple comparisons test. *Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: SAA1–mediated IL-1β release from MDSCs enhances SAA1 expression in ovarian cancer via the IL-1β/IL-1R/NF-κB axis. ( A ) Cytokine expression profile in MDSCs following SAA1 stimulation, detected by qRT-PCR. Data are presented as mean ± SEM from five independent experiments; unpaired two-tailed Student’s t-test. ( B ) Correlation analysis between SAA1 and IL1B expression in ovarian cancer using the TIMER database; Spearman’s rank correlation test. ( C ) IL-1β expression and secretion in MDSCs after SAA1 treatment across time points (0, 6, 12, 24 h), detected by qRT-PCR (left) and ELISA (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( D ) IL-1β expression and secretion in MDSCs after adding the TLR2/4 inhibitor SsnB to SAA1 treatment, detected by qRT-PCR (left) and ELISA (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( E ) SAA1 expression and secretion in SKOV3 cells after IL-1β treatment across time points (0, 6, 12, 24 h), detected by qRT-PCR (left) and ELISA (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( F ) Detection of SAA1 protein expression in SKOV3 cells after IL-1β treatment across time points (0, 6, 12, 24 h) by Western blotting (left) and densitometric analysis (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( G ) Prediction of transcription factors binding to the SAA1 promoter using TFDB, GTRD, and JASPAR databases. ( H ) Expression of predicted transcription factors in SKOV3 cells with or without IL-1β treatment, detected by qRT-PCR. Data are presented as mean ± SEM from three independent experiments; unpaired two-tailed Student’s t-tests. ( I ) SAA1 expression and secretion in SKOV3 cells after adding the NF-κB inhibitor APDC to IL-1β treatment, detected by qRT-PCR (left) and ELISA (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( J ) SAA1 expression and secretion in SKOV3 cells after adding the IL-1 receptor antagonist (IL-1RA) to IL-1β treatment, detected by qRT-PCR (left) and ELISA (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( K ) Detection of NF-κB signaling molecules in SKOV3 cells after adding APDC to IL-1β treatment by Western blotting (left) and densitometric analysis (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( L ) Detection of NF-κB signaling molecules in SKOV3 cells after adding IL-1RA to IL-1β treatment by Western blotting (left) and densitometric analysis (right). Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( M ) ChIP assay to detect the binding of transcription factor P65 to the SAA1 promoter in SKOV3 cells. Data are presented as mean ± SEM from three independent experiments; one-way ANOVA followed by Dunnett’s multiple comparisons test. ( N ) IL1B expression in ovarian cancer tissues based on the GEPIA database. Each dot represents one sample; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( O ) Association between IL1B expression and FIGO stage in ovarian cancer based on the GSE51088 dataset. Each dot represents one sample; Mann–Whitney U test. ( P ) Association between IL1B expression and histological grade in ovarian cancer based on the GSE63885 dataset. Each dot represents one sample; Kruskal–Wallis test. ( Q ) Detection of IL1B mRNA expression in benign and malignant ovarian tumor tissues by qRT-PCR. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. ( R ) Detection of IL-1β protein levels in serum from patients with benign and malignant ovarian tumors by ELISA. Each dot represents an individual patient; data are presented as mean ± SEM; Mann–Whitney U test. *Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor cells promote immunosuppression in ovarian cancer via a positive feedback loop with MDSCs through the SAA1–IL-1β axis

doi: 10.1186/s13046-025-03536-y

Figure Lengend Snippet: Deciphering the impact of SAA1-driven immunosuppression and its clinical relevance in ovarian cancer. ( A ) Abdominal circumference of C57BL/6 mice measured weekly for 4 weeks after intraperitoneal injection of SAA1-WT or SAA1-KO ID8 cells. Data are presented as mean ± SEM; two-way ANOVA. ( B ) Abdominal circumference at Week 4. Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( C ) Representative gross images showing differences in tumor nodule formation on the abdominal wall between SAA1-WT and SAA1-KO mice. ( D–E ) Detection of splenic G-MDSCs and M-MDSCs in SAA1-WT and SAA1-KO mice by flow cytometry ( D ) and quantification ( E ). Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-tests. ( F–G ) Detection of ascitic G-MDSCs and M-MDSCs in SAA1-WT and SAA1-KO mice by flow cytometry ( F ) and quantification ( G ). Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-tests. ( H ) Flow cytometry analysis of the number of Treg cells in the spleen and ascites of SAA1-WT and SAA1-KO mice. Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( I ) Flow cytometry to detect the ability of CD8⁺ T cells to secrete IFN-γ in the spleen and ascites of SAA1-WT and SAA1-KO mice. Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( J ) Flow cytometry to detect the ability of CD8⁺ T cells to secrete GZMB in the spleen and ascites of SAA1-WT and SAA1-KO mice. Each dot represents one mouse; data are presented as mean ± SEM; unpaired two-tailed Student’s t-test. ( K ) Representative multiplex immunofluorescence (mIF) images of tumor tissues from ovarian cancer patients with high or low SAA1 expression, with three patients in each group. ( L ) Correlation between SAA1 and CD33 expression in the GSE17260 , GSE32062 , and GSE51088 datasets; Spearman’s rank correlation test. ( M ) Correlation between SAA1 and IL1B expression in the GSE17260 , GSE32062 , and GSE51088 datasets; Spearman’s rank correlation test. ( N ) Kaplan–Meier analysis evaluating the prognostic value of SAA1 high CD33 high versus SAA1 low CD33 low expression for OS in TCGA-OC patients; log-rank test. ( O ) Kaplan–Meier survival analysis evaluating the prognostic value of SAA1 high IL1B high versus SAA1 low IL1B low expression for OS in TCGA-OC patients; log-rank test. ( P ) Kaplan–Meier survival analysis evaluating the prognostic value of the SAA1/IL1B/CD33 axis for OS in TCGA-OC patients; log-rank test. ( Q ) A nomogram constructed based on SAA1, IL1B, and CD33 expression from TCGA-OC data. ( R ) Schematic diagram showing how ovarian cancer cells form a positive feedback loop with MDSCs through the SAA1-IL-1β axis, promoting immune evasion. *Statistical significance: * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant

Article Snippet: The recombinant protein SAA1 (HY- P70510 , human; HY-P700309, mouse; MedChemExpress) was used at a final concentration of 200 ng/ml, and the TLR2/4 inhibitor Sparstolonin B (SsnB; HY-116213, MedChemExpress) was used at a final concentration of 20 μM.

Techniques: Injection, Two Tailed Test, Flow Cytometry, Multiplex Assay, Immunofluorescence, Expressing, Construct

Journal: Frontiers in Endocrinology

Article Title: Acute-serum amyloid A and A-SAA-derived peptides as formyl peptide receptor (FPR) 2 ligands

doi: 10.3389/fendo.2023.1119227

Figure Lengend Snippet: Amino acid sequence of naturally occurring and commercially available recombinant human A-SAA preparations discussed in the present paper.

Article Snippet: Eukarytoic recombinant human SAA1.1 (erhSAA1.1; Origene) , RS FFSFL GEAFD GARDM WRAYS DMREA NYIGS DKYFH ARGNY DAAKR GPGGV WAAEA ISDAR ENIQR FFGHG AEDSL ADQAA NEWGR SGKDP NHFRP AGLPE KYEQK LISEE DL.

Techniques: Sequencing, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: SAA1/FPR2 signaling between keratinocytes and neutrophils sustains chronic inflammation in Sweet syndrome

doi: 10.1172/JCI193566

Figure Lengend Snippet: ( A ) Ligand-receptor analysis reveals keratinocyte- and neutrophil-specific interactions. Keratinocytes expressed SAA1 transcripts and neutrophils expressed the FPR2 receptor (red dot, right-most column). ( B ) Dot plot demonstrating predominantly cell-specific expression of SAA1 and FPR2 transcripts. The dot size reflects the percentage of cells expressing the gene, and the color illustrates the level of gene expression. ( C ) Representative immunofluorescence staining images and quantification from 5 diseased and 5 control samples, confirming the expression of SAA1 and FPR2 in keratinocytes and neutrophils, respectively. Scale bars: 100 μm. ( D ) Dot plot comparing keratinocyte SAA1 and the control gene DEFB1 in different inflammatory skin conditions. The dot size reflects the percentage of cells expressing the gene, and the color illustrates the level of gene expression. ( E ) SAA1 secretion measured by ELISA in healthy neutrophils or healthy neutrophils exposed to keratinocytes. ( F ) Antibodies blocking SAA1 and FPR2 restored long-lived neutrophil lifespan to WT neutrophil levels. ( G ) Recombinant human SAA1 increased neutrophil survival at 72 hours ( n = 3 independent donors). Data indicate the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( E and G ) and 1-way ANOVA with individual comparisons ( F ).

Article Snippet: Freshly isolated neutrophils from healthy donors were treated with recombinant human SAA1 (SAA1-257H, Creative Biomart).

Techniques: Expressing, Gene Expression, Immunofluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, Blocking Assay, Recombinant

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Human SAA and/or HDL proteins compared in this study †

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Sequencing, Recombinant

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Binding of SAA preparations to influenza A virus (IAV)—Binding of SAAs to the Phil82 strain of IAV was tested by ELISA as described in Section 2 (“Materials And Methods”) using an anti‐serum amyloid A (anti‐SAA1) antibody (panel A). Binding of SAA1 was also tested in presence of maltose (panel B) and using an anti‐DDK antibody for detection (SAA1 has a DDK tail) (panel C). HDL binding to IAV was tested as well (panel D). * = P < 0.05 and ** = P < 0.01 compared to control. Results represent mean ± sem for 3 to 5 experiments

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Binding Assay, Virus, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Neutralization of influenza A virus (IAV) by serum SAA, but not recombinant, SAA preparations—The indicated strains of IAV were pre‐incubated with SAA and or HDL preparations and then these were used to infection MDCK cell monolayers as described in Section 2 (“Materials And Methods”). Serum SAA from Abcam (panel A), SAA complexed with HDL (SAAHDL; panel B), serum HDL (panel C), and two recombinant serum amyloid A (SAA1) preparations were compared (panel D; HDL and SAAHDL included for comparison). * = P < 0.05 and ** = P < 0.01 and *** = < 0.001 compared to control. Results represent mean ± sem for 3 to 5 experiments

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Neutralization, Virus, Recombinant, Incubation, Infection, Comparison, Control

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Stimulation of neutrophil hydrogen peroxide production by SAA and or HDL—Neutrophil H 2 O 2 generation was measured through decline in scopoletin fluorescence as described in Section 2 (“Materials And Methods”). Influenza A virus (IAV; Phil82 strain) was pre‐incubated with the indicated concentrations of SAA and/or HDL preparations and added to suspensions of neutrophils at time 0. Results of unstimulated cells (PBS) are included for comparison. Instances where serum amyloid A (SAA1) proteins increased IAV‐induced H 2 O 2 production are noted by * or ** in in legend, where * = P < 0.05 and ** = P < 0.01 compared to control. Results represent mean ± sem for 3 to 5 experiments

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Fluorescence, Virus, Incubation, Comparison, Control

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Effect of SAA and or HDL on neutrophil IL‐8 production or caspase activity—In panel A, IL‐8 production by neutrophils was measured by ELISA as described in Section 2 (“Materials And Methods”). ** = P < 0.01 compared to control. Results represent mean ± sem for 3 to 5 experiments. Panel B shows IL‐8 production in response to influenza A virus (IAV) alone or IAV combined with Apo‐serum amyloid A (Apo‐SAA1). Panel C shows caspase 3 activation by IAV alone or IAV combined with Apo‐SAA1, SAAHDL (SAA complexed with HDL), SAA1, or HDL. * = P < 0.05 compared to results with neutrophils in media alone. Results are mean ± sem of 4 experiments

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control, Virus, Activation Assay

Journal: Journal of Leukocyte Biology

Article Title: Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils

doi: 10.1002/JLB.4AB0220-116RR

Figure Lengend Snippet: Effects of TLR2 blocking antibodies, pertussis toxin (PT), and wortmannin of neutrophil responses to Apo‐serum amyloid A (Apo‐SAA1)

Article Snippet: We tested several commercially available forms of SAA1, including native human serum SAA complexed with HDL (SAAHDL; Fisher Scientific), SAA (19‐94) isolated from human serum (Abcam; Cambridge, United Kingdom, Abcam.com), recombinant SAA1 derived from E. coli (Peprotech; Rocky Hill, NJ, USA), and recombinant SAA1 derived from HEK cells (Origene, Rockville, MD, USA).

Techniques: Blocking Assay, Control, Virus

Journal: Cell metabolism

Article Title: Cytoarchitectural multi-depot profiling reveals immune-metabolic crosstalk in human colon-associated adipose tissue

doi: 10.1016/j.cmet.2025.12.008

Figure Lengend Snippet: (A) Cell-cell communication analysis of SAA signaling pathway for the indicated depots and cells. Line colors and widths represent the sending cell and the strength of the signal, respectively. (B) Pseudobulk comparison of differentially expressed genes in epiploic vs. subcutaneous depots, highlighting the indicated signaling pathways. (C) Expression of SAA1 mRNA upon stimulation with LPS (10 ng/mL), TNF-α (2.5 ng/mL), IL-1β (10 ng/mL), or IL-6 (10 ng/mL) for 24 h. Data are presented as fold change (FC) over vehicle. Significant (<0.05) p values compared with the vehicle from one-way ANOVA are shown. (D) Relative expression of SAA1 (left) and SAA2 (right) mRNA in adipocytes, ASPCs, adipose-derived endothelial cells (adECs), and THP1-derived macrophages (THP1 M0). Significant (<0.05) p values compared with the cell-type-specific control from one-way ANOVA are shown. (E) Results from ELISA measurements of SAA in conditioned media from human adipocytes incubated without or with LPS for 72 h. Results are displayed as mean, error bars represent standard error of the mean, and statistical significance was calculated using Student’s t test. (F) Analyses of DNA methylation in the indicated cell classes for the promoter and gene body regions of SAA1 and SAA2 , respectively. Chromosomal localization and motifs for NFKB2/RELA/REL and STAT3 are indicated for both genes. (G) SAA1 and SAA2 mRNA expression in adipocytes treated with or without LPS in the presence or absence of different inhibitors described in the main text. Data are presented as FC over vehicle. Significant (<0.05) p values compared with the vehicle from one-way ANOVA are shown. For (C), (D), and (G), data are displayed as geometric mean ± 95% confidence intervals with technical replicates displayed from three independent experiments. ASPC, adipose stromal and progenitor cell.

Article Snippet: Human recombinant SAA1 protein , OriGene , Cat#TP310664.

Techniques: Comparison, Protein-Protein interactions, Expressing, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Incubation, DNA Methylation Assay

Journal: Cell metabolism

Article Title: Cytoarchitectural multi-depot profiling reveals immune-metabolic crosstalk in human colon-associated adipose tissue

doi: 10.1016/j.cmet.2025.12.008

Figure Lengend Snippet: (A) Top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways based on differentially expressed genes from comparisons of SAA1- or LPS-treated vs. control-treated adipose-derived stromal-vascular cells. (B) Network view of the six common SAA1- and LPS-regulated pathways showing shared and treatment-specific genes. (C) Expression scores for SAA1- (left) and LPS-responsive (right) genes following a 6-h incubation with either ligand in stromal-vascular cells from subcutaneous human WAT. Results are displayed on the uniform manifold approximation and projection panel from and violin plots for each indicated cell class. Note that adipocytes and mesothelial cells were removed as these are not present in the stromal-vascular fraction from the subcutaneous depot. (D) Representative immunostaining of epiploic adipose tissue using antibodies directed against markers for Adipo SAA (CES1) and macrophages (CD68). Hoechst33342 was used as the counterstain for nuclei. Scale bar, 100 μm. ASPC, adipose stromal and progenitor cell; NES, normalized enrichment score.

Article Snippet: Human recombinant SAA1 protein , OriGene , Cat#TP310664.

Techniques: Control, Derivative Assay, Expressing, Incubation, Immunostaining

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased SAA3 expression and decreased femoral BMD in WT mice. WT and SAA3 KO male mice (3.5 mo old) were infused with vehicle (VEH) or PTH (40 µg/kg/d) for 12 d. (a) Saa3 mRNA expression in tibiae of WT mice. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by quantitative real-time PCR (qPCR) and data were reported as RQ values. (b) SAA3 protein in serum of WT and SAA3 KO mice by ELISA. Und, undetectable. (c) Change in in vivo femoral BMD calculated as: (BMD at end of infusion − BMD at start of infusion)/BMD at start of infusion. (d) Saa1, Saa2/Saa1, and (e) Ptgs2 (COX2) mRNA in WT and KO tibiae. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. mRNA expression was measured by qPCR and data were reported as RQ values. Bars are means ± SEM for n = 6 mice per genotype and treatment group. For (a), **P < 0.01, determined by two-tailed, unpaired t test. For (c) and (e), **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vivo, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: PTH infusion increased trabecular bone formation parameters only in SAA3 KO mice but increased resorption parameters similarly in both WT and SAA3 KO mice. WT and SAA3 KO male mice were infused with vehicle (VEH) or PTH. (a) Static histomorphometry of trabecular bone in distal femurs for Ob.S/BS, Oc.S/BS, and eroded surface per bone surface (ES/BS). (b) Representative TRAP-stained (red) and counterstained hematoxylin ×200 original magnification (scale bars, 100 µm) microscopic images of trabecular bone in distal femurs. Plump cuboidal osteoblasts around trabeculae are marked with yellow arrows. (c) Dynamic histomorphometry of trabecular bone in distal femurs for MS/BS, MAR, and BFR/BS. (c) Representative calcein-labeled (green) and demeclocycline-labeled (orange-brown) ×400 original magnification (scale bars, 25 µm) microscopic images of trabeculae. (e and f) Measurement of (e) serum bone formation markers, PINP, and BGLAP (osteocalcin) and (f) serum bone resorption markers, CTX and TRAcP5b, measured by ELISA. (g and h) mRNA expression of Runx2, Bglap (osteocalcin), Tnfsf11 (RANKL), and Tnfrsf11b (OPG) in the tibiae measured by quantitative real-time PCR and reported as RQ values. Both ends of each tibia were cut off to remove the growth plates and the marrow was not flushed. Bars are means ± SEM for n = 6 mice per genotype and treatment group. *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Staining, Labeling, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: Continuous PTH increased osteoblast differentiation in hBMSCs only when COX2 or RANKL activity was blocked. hBMSCs were cultured with vehicle (VEH) or PTH (10 nM), with or without NS398 (100 nM), a selective inhibitor of COX2, or OPG (100 ng/mL), which blocks RANKL binding to its receptor. (a) Measurement of accumulated medium PGE2 by ELISA. (b) Markers of osteoblast differentiation, ALPL (alkaline phosphatase) and BGLAP (osteocalcin), and alizarin red staining for mineralization at day 21. (c) TNFSF11 (RANKL) mRNA at day 7 and TRAP staining at day 8. Scale bar, 100 µm. (d) BGLAP mRNA and alizarin red staining at day 21. (e) RUNX2 mRNA at day 7 and IGF1 and BMP2 mRNA at day 21. (f) WNT10B and DKK1 mRNA at day 7. mRNA was measured by quantitative real-time PCR and data are reported as RQ values. Bars are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (c), **P < 0.01, determined by two-tailed unpaired t test. For (d)–(f), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. Und, undetectable.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Activity Assay, Cell Culture, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Endocrinology

Article Title: Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A

doi: 10.1210/en.2018-00265

Figure Lengend Snippet: NS398 blocked the RANKL induction of SAA1 and SAA2 expression in hBMMs but did not affect osteoclast-like cell numbers, and SAA1 and SAA2 inhibited PTH-stimulated osteoblastic differentiation in hBMSCs. (a–c) hBMMs were treated with M-CSF (30 ng/mL) plus vehicle (VEH) or M-CSF plus RANKL (30 ng/mL each) with/without NS398 (100 nM). (a) SAA1 and SAA2 protein measurement in the culture medium of hBMMs measured by ELISA. (b) SAA1 and SAA2 mRNA at day 3. (c) TRAP-stained microscopic images at ×100 original magnification (scale bars, 200 µm) and TRAP+ MNC counts per well. (d) ALPL (alkaline phosphatase) and BGLAP (osteocalcin) mRNA expression in hBMSCs at day 21 treated with VEH or PTH in presence of OPG (100 ng/mL) to prevent osteoclastogenesis with/without rhSAA1 (10 µg/mL) or rhSAA2 (10 to 50 ng/mL). Data are means ± SEM for n = 3 independent samples. For (a), **P < 0.01, significantly different from VEH treated at same time point, determined by two-tailed unpaired t test. For (c), **P < 0.01, significantly different from day 4, determined by one-way ANOVA, post hoc Bonferroni pairwise multiple comparisons. For (b) and (d), *P < 0.05, **P < 0.01, determined by two-way ANOVA, post hoc Bonferroni pairwise multiple comparisons.

Article Snippet: SAA1 protein accumulated in the medium of human BMMs was measured by an SAA1 human ELISA kit (KA2108; Abnova Corporation, Walnut, CA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test