Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 1 SAA1 is overexpressed in response to pressure overload. (A and B) Representative cardiac micrographs of HE staining and quantification analysis of sham group and TAB mice. Magnification, 200×. Scale bar, 50 μm. (C and D) Heart weight/body weight and heart weight/tibia length ratios of mice after sham or TAB operation. (E) Typical micrographs of hematoxylin and eosin and Masson's trichrome staining of sham group and TAB mice. (F–H) Western blotting and PCR of SAA1 in the sham group and transverse aortic banding group. (I) Immunohistochemical image of SAA1 expression in the sham group and the 8-week transverse aortic banding group. TAB indicates transverse aortic banding; TAB 8W, 8-week transverse aortic banding group. *p < .05 compared with the Sham group.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Staining, Western Blot, Immunohistochemical staining, Expressing

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 2 SAA1 knockout relieves cardiac dysfunction after pressure overload. (A) Characteristic echocardiographic images for each group. (B-D) Statistical results for the ejection fraction, left ventricular fractional shortening, and heart rate in each group (n = 12). (E-G) Results for the cardiac output, maximal rate of pressure development (dP/dtmax), and maximal rate of pressure decay (dP/dtmin) in each group (n = 12). TAB indicates transverse aortic banding; CO, cardiac output; dp/dtmax, maximal rate of left ventricle pressure development; dp/dtmin, minimal rate of left ventricle pressure development; EF, ejection fraction; FS, fractional shortening; and HR, heart rate. *P < 0.05 compared with Sham group.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Knock-Out

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 3 SAA1 absence does not relieve transverse aortic banding-induced cardiac hypertrophy. (A) The picture of the heart in each group. (B and C) Heart weight/body weight and heart weight/tibia length ratios of wild-type (WT) and SAA1-knockout mice (KO) after sham or transverse aortic banding (TAB) operation. (D and E) Representative hematoxylin detection of WT or SAA1-KO mice after sham or AB operation shows SAA1 deficiency does not improve pressure overloaded cardiac hypertrophy. (F, G, K) The mRNA expression of ANP, BNP andβ-MHC in each group (n = 6). (H–J) Representative blots and quantitative results for ANP and β-MHC protein expression in myocardium in each group (n = 6). TAB indicates transverse aortic banding; ANP indicates atrial natriureticpeptide; BNP, B-type natriuretic peptide.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Knock-Out, Expressing

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 4 SAA1 deficiency lessens transverse aortic banding-induced cardiac fibrosis. (A) Typical image of the heart with Picrosirius staining. scale bar: 50 μm. (B) Quantification of the total collagen volume in the indicated group (n = 6). (C–E) PCR analysis of fibrotic markers (TGF-β, αSMA, collagen Iα) (n = 6). (F–J) Representative blots and quantitative results for Col I, collagen IV and Fibronectin, and E-Cadherin protein expression in the myocardium in each group (n = 6). TAB indicates aortic banding; Col1α, collagen Iα; Col3, collagen type 3; KO, knockout mice; PSR, Picrosirius red; TGF-β, transforming growth factor-β; WT, wild-type; αSMA, alpha-smooth muscle actin. *p < .05 compared with the corresponding wild type transverse aortic banding group.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Staining, Expressing, Knock-Out

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 5 Disruption of SAA1 alleviates inflammation in aortic banding-induced heart. (A and B) Western blotting analysis of protein expressions of p65, p-p65, IkBα, p-IkBα, p38, p-p38, ERK, p-ERK, JNK, p-JNK inSAA1+/+, and SAA1−/− hearts with or without cardiac AB. GAPDH was used as a loading control. (C–G) Quantification of NF-κB/p38/JNK signaling pathway-related protein levels. *p < .05. TAB, transverse aortic banding.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Disruption, Western Blot, Control

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 6 SAA1 deficiency decreases cardiac fibrosis through inhibiting TGFβ/Smad signal pathway activation in mice after aortic banding. (A) Serum level of IL-6, IL-1β, TNFα from mice was detected using an ELISA. (B) Western blotting analysis of TGF-β, α-SMA, p-Smad2, and p-Smad3 and Smad7 proteins. GAPDH was used as a loading control. (C–G) Quantification analysis of the proteins' expressions. Data are presented as the mean ± SE, n = 6 mice per group.*p < .05.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 7 Overexpression of SAA1 aggravates the fibrotic response in vitro. (A) RT-qPCR analysis of mRNA level of SAA1 between cardiomyocytes and cardiac fibroblasts. The data were compared by unpaired Student t-test. (B) RT-qPCR analysis of the mRNA expression level of SAA1, ColIα, α-SMA when in cardiac fibroblasts were stimulated by TGF-β. (C and D) PCR and western blotting analysis of the SAA1 expression in control, pLV-Vector, and pLV-SAA1 groups. (E) Immunofluorescence staining of αSMA. (F–K) Representative western blotting and quantitative results for P-P65/T-P65, P-IκBα/T-IκBα, P-Smad2/T-Smad2, P-Smad3/T-Smad3, and TGF-βprotein expression in cardiac fibroblasts each group. CF, cardiac fibroblasts; CMs, Cardiomyocyte; *p < .05 versus control, #p < .05 versus pLV-Vector group, and p < .05 versus pLV-SAA1 group.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Western Blot, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: SAA1 deficiency alleviates cardiac remodeling by inhibiting NF‐κB /p38/ JNK and TGFβ /Smad pathways

doi: 10.1096/fj.202201506r

Figure Lengend Snippet: FIGURE 8 Knock-down SAA1 ameliorates the fibrotic response in vitro. (A–C) RT-qPCR analysis of mRNA level of ColIα, Col3, and α-SMA (n = 6). (D) RT-qPCR analysis of inflammatory cytokines IL-1β, IL-6, and TNF-α in the indicated groups (n = 6). (E–K) Representative blots and quantitative results for SAA1, P-P65/T-P65, P-IκBα/T-IκBα, P-Smad2/T-Smad2, P-Smad3//T-Smad3, and TGF-βprotein expression in cardiac fibroblasts each group. *p < .05.

Article Snippet: To determine the impact of SAA1 on cardiac remodeling, SAA1 knockout (KO) mice were generated and purchased from Cyagen Biosciences (Supplementary material 2).

Techniques: Knockdown, In Vitro, Quantitative RT-PCR, Expressing

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: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Primer sequences used for quantitative PCR

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Radiation time response in mouse SAA1 measured using ELISA in 8 Gy irradiated female C57BL/6J mice at 0, 1, 2, 4, and 6 hours post-irradiation. n=3 per group. *, P<0.05 in the irradiated mice compared with control mice.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Time and dose response of SAA1 after TBI. (A) SAA1 was measured using ELISA in 0, 1, 2, 4, 8 and 12 Gy irradiated female C57BL/6J mice at 0.125, 0.5, 1, 2, 3, 5 and 7 days post-irradiation. SAA1 dose-dependent change at (B) 0.25, (C) 0.5, (D) 1, (E) 2, (F) 3, (G) 5 and (H) 7 days after exposure to radiation. Each black dot represents one animal. Error bars indicate ± 1 SD for each radiation exposure group. n=8 per group (n=7 at 7 days after 8 Gy, n=2 at 7 days after 12 Gy). SAA1, serum amyloid A1; TBI, total body irradiation.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: P values associated with two-tailed Student’s t -test for SAA1 concentrations after comparing control and irradiated groups

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: SAA1 mRNA time-dependent increase after radiation in (A) liver, (B) lung, (C) thymus, (D) spleen, (E) bone marrow and (F) small intestine measured using Quantitative PCR in control and 8 Gy irradiated female C57BL/6J mice at 0.125, 0.5, 1, 2, 3, 5 and 7 days post-irradiation. Error bars indicate ± 1 SD for each radiation exposure group. n=6 to 8 per group. *, P<0.05, *, P<0.01, ***, P<0.001, and ****, P<0.0001 in the irradiated mice compared with the control mice. SAA1, serum amyloid A1.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: SAA1 expression and systemic infection parameters in the same mice. (A) SAA1 and LPS in serum and 16S rRNA in the liver of the same animals measured on 31 mice on day 0,1, 5 and 7 (7 mice on day 7, and 8 per group on day 0, 1 and 5) after exposure to 8 Gy TBI. (B) SAA1 and PCT in serum in the same animals was on 23 mice on days 0, 0.125, 0.5, 1, 2, 3, 5 and 7 (2 mice on days 7 and 3 per group on days 0, 0.125, 0.5, 1, 2, 3 and 5) after exposure to 12 Gy TBI. SAA1, serum amyloid A1; TBI, total body irradiation.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Multiple linear regression analysis between serum SAA1 concentration and number of lymphocyte and irradiation dose

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: SAA1 cut-off values of classification within 2 days post-irradiation

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques:

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: PBI patterns and SAA1 response. (A) The liver of C57BL/6J mice was shielded in PBI-1 and PBI-3 and in the corresponding groups PBI-2 and PBI-4 the liver was exposed. (B) SAA1 concentration in serum and (C) mRNA expression in liver of control, total-body irradiation and PBI group at 12 hours after 8 Gy irradiation. Error bars indicate ±1 SD for each radiation exposure group. n=6 per group. **, P<0.01, ***, P<0.001, and ****, P<0.0001 in the irradiated mice compared with the control mice. PBI, partial body irradiation; SAA1, serum amyloid A1.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Dynamic SAA1 concentration and corresponding date of death within 30 days after 10 Gy irradiation

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Mean and standard deviation (SD) of SAA1 concentration in 92 healthy mice

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Standard Deviation, Concentration Assay

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Kaplan-Meier survival curves of mice. P value determined by log-rank test. Serum SAA1 in mice treated with amifostine before 10 Gy irradiation and in the 10 Gy group on day −4, 1, 3, 5 and 7. n=8 in 10 Gy + Amifostine group, n=20 in 10 Gy group. ***, P<0.001. SAA1, serum amyloid A1.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: SAA1 concentration in the Amifostine group and corresponding death date within 30 days

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Irradiation

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Clinical parameters and corresponding SAA1 concentration in all 17 NPC patients

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Annals of Translational Medicine

Article Title: Serum amyloid A1 as a biomarker for radiation dose estimation and lethality prediction in irradiated mouse

doi: 10.21037/atm.2019.12.27

Figure Lengend Snippet: Effect of radiotherapy on serum SAA1 in nasopharyngeal carcinoma patients. (A) The scatter plot shows SAA1 concentration before radiotherapy and corresponding expression after radiotherapy in 17 patients with nasopharyngeal carcinoma (****, P<0.0001). (B) ROC curve of SAA1 as a biomarker for predicting radiation exposure in patients with nasopharyngeal carcinoma. SAA1, serum amyloid A1.

Article Snippet: Serum proteins such as SAA1 and procalcitonin (PCT) were measured using enzyme-linked immunosorbent assay (ELISA) kits (Mouse SAA1: EK1190, Boster, Wuhan, China; Human SAA1: EK1544, Boster, Wuhan, China; PCT: E10371m, CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Biomarker Assay

Journal: Nature Communications

Article Title: Multi-omics with dynamic network biomarker algorithm prefigures organ-specific metastasis of lung adenocarcinoma

doi: 10.1038/s41467-024-53849-3

Figure Lengend Snippet: a Serum DNB proteins were overlapped with primary lesion DNB genes, in which comparative analyses were made to filter the most relevant organ-specific serum biomarkers ((1) and (2)). Different letter marks indicate significant differences ( P < 0.05). In this case, proteins/genes marked in red indicate high specificity in their belonging groups (five different metastatic states). Kruskal–Wallis and Dunn’s tests were performed to make inter-group comparisons. b SAA1 expression distribution in the UMAP plot. c The clinical significance of SAA1 as a biomarker in lung adenocarcinoma was illustrated, in which the overall survival and first progression results were significant. d Schematic figure indicating animal model validation of SAA1 as a serum biomarker for bone metastasis. The number of mice in each group was 16. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. e The serum SAA1 was measured in two different cell lines-generated bone metastasis models. A total of 16 mice were included after model construction for each cell line (Lewis lung carcinoma (LLC)-model or KRAS G12D TP53 −/− (KP)-model), and a comparison was made between primary ( n = 8) and bone metastasis ( n = 8) groups. The serum level of SAA1 was significantly higher in the samples of bone metastasis compared those without bone metastasis. One-way ANOVA was performed to make inter-group comparison. The P value for LLC-model comparison between primary and bone metastasis groups was 0.005, and the P value for KP-model comparison between primary and bone metastasis groups was <0.001. Source data are provided as a Source Data file.

Article Snippet: The information of the antibodies we used was listed as follows: (i) EEF2 Polyclonal antibody (Proteintech, 20107-1-AP, dilution 1:1000); (ii) RPS3A Polyclonal antibody (Proteintech, 14123-1-AP, dilution 1:1000); (iii) SAA1 Polyclonal antibody (Proteintech, 16721-1-AP, dilution 1:1000); (iv) 14-3-3E Monoclonal antibody (Proteintech, 66946-1-Ig, dilution 1:3000); (v) PSMB6 Polyclonal antibody (Proteintech, 11684-2-AP, dilution 1:1500).

Techniques: Expressing, Biomarker Discovery, Animal Model, Generated, Comparison

Journal: Nature Communications

Article Title: Multi-omics with dynamic network biomarker algorithm prefigures organ-specific metastasis of lung adenocarcinoma

doi: 10.1038/s41467-024-53849-3

Figure Lengend Snippet: a Schematic figure validating the role in lung cancer bone metastasis clinically. For the first validation cohort, three paired lung adenocarcinoma samples of primary and bone metastatic lesions were collected and analyzed through scRNA-seq. For the second validation cohort, 20 lung adenocarcinoma bone metastatic lesions were collected, and eight of them were analyzed through bulk RNA-seq and compared with the normal bone tissues from healthy controls, while IHC staining was conducted on the remaining 12 samples to confirm the expression of SAA1, YWHAE, EEF2, PSMB6, and RPS3A. Created in BioRender. Wen, Y. (2024) BioRender.com/c79s395. b UMAP plot demonstrating 19 cancer cell clusters. c UMAP plot of cancer cells, grouped by cell origin. d The major cell cluster constituents in lung or bone metastatic samples. e The expression percentage of SAA1 in each cancer cell cluster. f The variation of expression percentage of SAA1 in each cancer cell cluster and total cancer cell clusters from primary to bone metastatic lesions. g The heatmaps illustrated that many of the DNB genes prefiguring bone metastasis, including SAA1 , were highly enriched in cancer cell clusters of bone metastatic lesions. h For the second validation cohort, the expression Log 2 foldchange of the DNB genes indicating different metastases was examined, and SAA1 was the only gene denoting a Log 2 foldchange of ~10 times between bone metastatic lesions and normal bone tissues. i The IHC staining results of SAA1 (1), YWHAE (2) PSMB6 (3), RPS3A (4), and EEF2 (5) in bone metastatic lesions in the second validation cohort and their corresponding statistical analysis (6), with n = 12 in each group. The data were presented as mean ± SD; P < 0.001 for the comparison between SAA1 and others. One-way ANOVA was performed to make inter-group comparison. j The serum level of SAA1 in groups of healthy controls ( n = 30), bone ( n = 10), "Brainplus" ( n = 14), lung ( n = 12), pleura ( n = 11); P value for inter-group comparison was listed as follows: bone-healthy volunteers: P = 0.01; bone-Brainplus: P = 0.008; bone-lung: P = 0.005; bone-pleura: P = 0.005 (1); and YWHAE in groups of bone ( n = 10), brainplus (n = 12), lung ( n = 12), pleura ( n = 11) were comparatively analyzed, with P value for inter-group comparison was listed as follows: bone-Brainplus: P = 0.305; bone-lung: P = 0.860; bone-pleura: P = 1.000 (2). The data were presented as mean ± SD. One-way ANOVA and Dunnett’s test corrections were performed to make inter-group comparison. * P < 0.05; ** P < 0.01; *** P < 0.001; ns: not significant. Bone mets.: Bone metastasis.

Article Snippet: The information of the antibodies we used was listed as follows: (i) EEF2 Polyclonal antibody (Proteintech, 20107-1-AP, dilution 1:1000); (ii) RPS3A Polyclonal antibody (Proteintech, 14123-1-AP, dilution 1:1000); (iii) SAA1 Polyclonal antibody (Proteintech, 16721-1-AP, dilution 1:1000); (iv) 14-3-3E Monoclonal antibody (Proteintech, 66946-1-Ig, dilution 1:3000); (v) PSMB6 Polyclonal antibody (Proteintech, 11684-2-AP, dilution 1:1500).

Techniques: Biomarker Discovery, RNA Sequencing, Immunohistochemistry, Expressing, Comparison

Journal: Nature Communications

Article Title: Multi-omics with dynamic network biomarker algorithm prefigures organ-specific metastasis of lung adenocarcinoma

doi: 10.1038/s41467-024-53849-3

Figure Lengend Snippet: a Schematic figure of neural network establishment and data analysis workflow. External labeled validation single-cell datasets (primary tumor and bone metastasis) were used for validation of genomic trace of metastatic signature. For the training set, bone and “Boneplus” were combined as a bone metastasis group. Lung (intra-pulmonary) and pleural metastasis were combined as a lung metastasis group. The reason for the signature combination was due to limited consensus on the discrete metastatic classification of public datasets. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. b The receiver operating characteristic (ROC) curve indicating the effectiveness of the trained neural network. c The chart illustrating connecting information of verified group (left) and labeled metastatic status (right). d Transcriptomic signatures of primary tumor ( n = 1), brain metastasis ( n = 1) and bone metastasis ( n = 1) from the external dataset (GSE123902), in which SAA1 was in the top gene signature of bone metastasis. Source data are provided as a Source Data file.

Article Snippet: The information of the antibodies we used was listed as follows: (i) EEF2 Polyclonal antibody (Proteintech, 20107-1-AP, dilution 1:1000); (ii) RPS3A Polyclonal antibody (Proteintech, 14123-1-AP, dilution 1:1000); (iii) SAA1 Polyclonal antibody (Proteintech, 16721-1-AP, dilution 1:1000); (iv) 14-3-3E Monoclonal antibody (Proteintech, 66946-1-Ig, dilution 1:3000); (v) PSMB6 Polyclonal antibody (Proteintech, 11684-2-AP, dilution 1:1500).

Techniques: Labeling, Biomarker Discovery, Generated

Journal: Journal of Inflammation Research

Article Title: LPS-Induced Inflammation Affects Midazolam Clearance in Juvenile Mice in an Age-Dependent Manner

doi: 10.2147/JIR.S321492

Figure Lengend Snippet: Data Comparison Between Experimental and Control Groups of Mice

Article Snippet: SAA1 levels were measured by ELISA (Cusabio, CSB-EL020656MO, Wuhan, China) in plasma samples.

Techniques: Comparison, Control

Journal: Journal of Inflammation Research

Article Title: LPS-Induced Inflammation Affects Midazolam Clearance in Juvenile Mice in an Age-Dependent Manner

doi: 10.2147/JIR.S321492

Figure Lengend Snippet: Covariate Analysis

Article Snippet: SAA1 levels were measured by ELISA (Cusabio, CSB-EL020656MO, Wuhan, China) in plasma samples.

Techniques: Selection

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Model of allergen-induced airway hyperresponsiveness (AHR) for wild-type (WT) and Saa–/– mice (both C57BL/6 background). Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 1a-​-jj and Extended Data Fig. 2 and ​and3).3). (b) For SAA1 antibody blockade, we used an established mouse model of allergen-induced AHR sensitizing WT BALB/cJ mice i.t. on day 0 and 14 with 100 µg of HDM extract + isotype control, or HDM + αSAAab. Airway measurements and tissue harvests were performed 72 h after the last allergen challenge (used in Extended Data Fig. 4). (c) In short-term exposure protocols WT and Saa–/– mice (both C57BL/6 background) received a single HDM challenge (100 μg) were sacrificed 16 h later (used in Fig. 2a-​-d).d). (d) In short-term exposure protocols BALB/cJ mice received a single HDM challenge (100 μg)+ isotype control, HDM challenge + HDL (200μg) and isotype control, or HDM + αSAAab and were sacrificed 24 h later (used in Fig. 2e). Contol mice received either PBS + isotype or PBS + HDL and isotype control. (e) For overexpression of SAA1 in vivo, mice were injected 20 µg of DNA complexed to polyethylenimine at day 0, exposed to PBS or HDM 48 h later and ILC2s as well as BAL cytokines measurements were performed on day 3 (used Fig. 2f). (f) WT and Saa–/– mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg with extracts from the parasitic worm Schistosoma mansoni (a Puerto Rican isolate). Tissues were harvested 72 h after the last allergen challenge (used in Fig. 4). (g) For FPR2 blockade (WRW4, 2 mg/kg), WT BALB/cJ mice were sensitized and challenged i.t. on day 0 and 14 with 100 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 7a-​-f).f). (h) In short term exposure experiments, BALB/cJ mice received a single HDM challenge (100 μg) or HDM + WRW4 and were sacrificed 24 h later (used in Fig. 7g and ​andi).i). (i) Model of Alternaria alternata (Alt a )-induced airway inflammation for WT and Saa–/– mice. Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 µg of Alt a extract. Tissues were harvested 72 h after the last allergen challenge (used in Extended Data Fig. 9f-​-jj).

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Over Expression, In Vivo, Injection

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 mRNA expression in nasal epithelial cells of CRS patients and matched control donors. (c) HDM (100 μg/ml)-triggered IL-33 concentration in primary nasal epithelial cells of CRS patients as compared to controls. (d) Dissociation of hexameric SAA was analyzed separating lipid-free and lipid-bound SAA by HDL pull down using a polyclonal goat antibody specific for human ApoA1 and immunoblotting with a monoclonal mouse antibody specific for human SAA1 (Acris). (e) Bar graph represents quantitative analysis of SAA monomer band intensities (LI-COR Image Studio Software). (f) SAA1 protein amounts in sera of control donors and HDM allergic individuals. Data represents means ± SEM of (a and b) n=16 control and n=27 CRS, (c) n=6 control and n=12 CRS patients per group, (e) n=5 control and n=8 CRS patients per group and (f) n=18 control and n=27 HDM allergic patients per group. Representative immunoblot of one control and one CRS patient (d). Cropped images are shown. P values were calculated with a two-tailed test using Student’s t-test with Welch’s correction. ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing, Concentration Assay, Western Blot, Software, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) AHR (*P < 0.0105), (b) total serum IgE concentrations (*P = 0.0265), (c) eosinophil infiltration into the lungs, and (d) PAS stained lung sections of isotype (iso), HDM+isotype (HDM), or HDM+αSAAab-treated (αSAA) WT BALB/c mice. Antibodies were administered at 5 μg/i.t. Frequency of (e) Lin-CD45+ST2+IL-13+ ILC2s (**P = 0.0027, ***P = 0.0002), (f) TH2 and (*P = 0.0260, ***P = 0.0001) (g) TH17 cells (*P = 0.0149, ***P = 0.0005) in the lungs of these mice. Data represents means ± SEM of pooled data from 2 independent experiments containing (a) n=6 PBS+iso, n=7 HDM+iso, n=9 HDM+αSAA animals per group; (b, c, f, g) n=9 PBS+iso, n=11 HDM+iso, n=13 HDM+αSAA animals per group or are representative of 2 independent experiments with (e) n=4 PBS+iso, n=5 HDM+iso, n=7 HDM+αSAA animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc analysis that compares HDM to iso and αSAA counterparts. ****P ≤ 0.0001

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Neutralization, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Increase of the type 2 cytokines (a) IL-33 (*P = 0.0138), (b) IL-25, and (c) TSLP in the BAL of WT and Saa–/– mice 24h after a single dose of PBS or HDM. (d) Numbers of IL-13+ ILC2 cells in the lungs of these mice. (e) Effects of local SAA1 neutralization in WT BALB/c mice through HDL (*P = 0.0117) or local SAA antibody blockade (αSAA, *P = 0.0392) of WT BALB/c receiving isotype (iso), HDM+isotype (HDM+iso), or HDM+αSAAab-treated (HDM+αSAA). (f) Effects of SAA1 overexpression on numbers of Lin-CD45+CD25+ST2+IL-13+ ILC2s in the lungs. For overexpression, WT mice were injected retro-orbitally with a SAA1 overexpression plasmid (SAA1, grey bars) or a non-coding control vector (pcDNA, open bars). Data represent means ± SEM of pooled data from 2 independent experiments containing (a-c) n= 6 WT PBS, n=8 WT HDM, n=7 Saa–/– PBS and n=10 Saa–/– HDM animals per group; (d) n= 5 WT PBS, n=9 WT HDM, n=6 Saa–/– PBS and n=9 Saa–/– HDM animals per group; (e) n=7 PBS+iso, n=6 PBS+iso/HDL, n= 11 HDM+iso, n=13 HDM+iso/HDL, n=12 HDM+αSAA animals per group; (f) n=3 pcDNA PBS, n=9 pcDNA HDM, n=3 SAA1 PBS and n=10 SAA1 HDM animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc analysis (a-e) or two-sided Student’s t-test (f). ***P ≤ 0.001

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Neutralization, Over Expression, Injection, Plasmid Preparation, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) HDM, rBlo t 13, rDer p2 or rDer p 23 were separated by SDS-PAGE followed by detection with rSAA1 and a mouse monoclonal antibody specific for human SAA1. (b) Migration of SAA1 (1 mg/ml) in PBS was analyzed in the presence of increasing amounts of the mite FABP Blo t 13 (4:1 (0.25 mg/ml), 2:1 (0.5 mg/ml), 1:1 (1 mg/ml) and 1:2 (2 mg/ml) of Blo t 13) by native PAGE followed by immunoblot analysis using mouse monoclonal antibody specific for human SAA1 (R&D systems; MAB30196). (c) SAA1 (1 mg/ml) was chemically cross-linked in the presence of Blo t 13 added at a ratio of 1:2 or 1:4 (as indicated) using 0.0025%, 0.005%, and 0.01% glutaraldehyde and analysed by immunoblot using sequence-specific (amino acid 14–30) rabbit antiserum for human SAA1 (densitometric analysis shown in supplementary table 1). (d) HDM-induced IL-33 concentrations in BEAS-2B cells after siRNA-mediated silencing of SAA1 (siSAA1) or non-targeting scrambled siRNA (siNT) (**P = 0.0002, ns = 0.1911). (e) IL-33 amounts induced by individual mite or unrelated major cat (Fel d 1) and birch pollen (Bet v 1) allergens (10 μg/ml) (. (f) Blo t 13-induced IL-33 in BEAS-2B cells with siRNA-mediated silencing of SAA1 (siSAA1) or transfected with non-targeting scrambled siRNA (siNT). (g) IL-33 amounts induced by Der p 13-depleted HDM extract and HDM extract where Der p 13 was neutralized (100 μg/ml). (h) BAL IL-33 levels (*P = 0.0209, ***P = 0.006) as well as (i) Lin-CD45+ST2+IL-13+ ILC2s in the lungs of WT BALB/c mice receiving a single i.t. challenge with 100 μg Der p 13-depleted HDM extract as compared to isotype-treated control extract (*P = 0.0423). IL-33 amounts induced in the BEAS-2B cell line and SAA1 binding by the human fatty acid binding proteins (j) FABP5 (**P = 0.0207) and (k) FABP7. Cropped images are shown. Data are shown as means ± SEM and are pooled data from 2 (d, e, g, j) or 3 (h, i) independent experiments or representative of 2 (k) or 3 independent experiments (f) each containing at least n=4 biologically independent samples or n=8 PBS, n=12 HDM-isotype and n= 14 HDM-α-group 13 (h) and or n=10 PBS, n=13 HDM-isotype and n= 14 HDM-α-group 13 (i) animals per group. Immunoblots are representative of an experimental n=2 (a-c, j, k). P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (d, e, f), Dunett’s (h) or Holm-Sidaks (i) post hoc analysis, and two-tailed Student’s t-test with Welch’s correction (g, j, k) ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: SDS Page, Migration, Clear Native PAGE, Western Blot, Sequencing, Transfection, Binding Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Migration of SAA1 (1 mg/ml) in IMDM media was analyzed in the presence of the mite FABP rBlo t 13 (1 mg/ml) by native PAGE followed by immunoblot analysis using a sequence-specific antiserum (amino acid 89–104) raised against human SAA1. (b) Effects of the protein synthesis inhibitor cycloheximide on IL-33 concentrations in BEAS-2B cells treated for 30 min with HDM (100 μg/ml). (c) Cell viability of BEAS-2B cells after HDM exposure over time as measured by continuous reduction of a cell viability substrate by viable cells (**P = 0.0041). SAA1 concentrations in cells after (d) siRNA-mediated silencing of SAA1 (siSAA1) or non-targeting scrambled siRNA (siNT). Effects of SAA1 on HDM-induced IL-6 (**P = 0.0086, ***P = 0.0007) and IL-8 release in cells with siRNA mediated knockdown of SAA1 (siSAA1) (e and f). (g) HDM-induced IL-6 amounts after Der p 13-depletion and/or neutralization. Data are shown as means ± SEM and are representative of 2 (b, e) or 3 (c) independent experiments or pooled data from 2 independent experiments (d, f) each containing at least n=4 biologically independent samples. Representative analysis of SAA1 migration patterns in the presence of Blo t 13 using sequence-specific rabbit antiserum raised against human SAA1 (aa 89–104) (a). IL-6 amounts were analysed for one representative experiments with n=5 biologically independent samples (g). Cropped images are shown. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) followed by Dunett’s post (b) or Tukey’s hoc analysis (e,f), two-way ANOVA followed by Dunnet correction (c) or Student’s t-test (d, g). ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Migration, Clear Native PAGE, Western Blot, Sequencing, Neutralization, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Sequence of SAA1. Indicated are: secondary structure α-helices (α 1–4) and loops (connecting lines between α-helices), C-terminal tail (CTL). Amino acids located within the hydrophobic core of the SAA1 hexamer are shaded in green. The epitopes recognized by affinity-purified IgGs raised against residues 27–44, 40–63, 68–84, and 89–104 are indicated by yellow lines (used in panel e). The mutation site at the hydrophobic core is indicated by an orange dot (used in panel f). C-terminal deletion of amino acids (Δ1–11) is shaded in blue. (b) BEAS-2B supernatants were cleared of lipid-bound SAA1 pulling down HDL-bound SAA1 using a polyclonal goat anti-ApoA1 antibody. Cleared supernatants were immunoblotted with a monoclonal mouse anti-human SAA1 antibody (Acris). (c) IL-33 secretion induced by rSAA1 alone (open bars) or in complex with a mouse monoclonal antibody specific for human SAA1 (αSAA ab, closed bars) in the BEAS-2B cell line. (d) Supernatants of cells left either untreated (media) or stimulated with recombinant SAA1 (rSAA) and immunoblotted with a monoclonal antibody specific for human SAA (R&D Systems; MAB30196). (e) IL-33 release from BEAS-2B cells after incubation with sequence-specific rabbit antisera specific for human SAA1 (**P = 0.0029). (f) HDM-triggered IL-33 release in BEAS-2B cells transfected with empty plasmid control (EV), wildtype SAA1 (WT) overexpression plasmid or a SAA1 plasmid with a Trp to Ala mutation at position 53 of the amino acid sequence (W53A) to mutate the hydrophobic core of SAA1 (WT to EV **P = 0.0032; WT to W53A **P = 0.0085). (g) HDM-induced IL-33 amounts in epithelial cells grown in media supplemented with charcoal-stripped FBS (**P = 0.0027). Cropped images are shown. Data shown as means ± SEM represent are representative of 2 independent experiments (c, d, f, g) or 3 (e) each containing at least n=4 biologically independent samples. Blots are representative of 2 (d) and 4 (b) independent experiments. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (e, g) or Dunett’s post hoc analysis (f). ****P ≤ 0.0001

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Sequencing, Affinity Purification, Mutagenesis, Recombinant, Incubation, Transfection, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) mRNA and (b) protein amounts of SAA in response to HDM (100 μg/ml). (c) SAA1 hexamer after rBlo t 13 stimulation was analyzed as described in Fig. 3b. (d) Bar graph represents quantitative analysis of SAA hexamer using LI-COR Image Studio Software. (e) Concentration-dependent IL-33 release from BEAS-2B cells induced by rBlo t 13. Data are shown as means ± SEM and are pooled data from 2 independent experiments (b, e) each containing n=4–5 replicate wells or representative of 2–3 independent experiments (d). SAA1 mRNA expression, normalized to the average of housekeeping genes, is presented as mean value ± SEM (n = 5). Immunoblot is representative of an experimental n=2. Cropped images are shown. P values were calculated with a two-tailed test using Student’s t-test (a), two-way analysis of variance (ANOVA) followed by Dunnet’s correction (b) or one-way ANOVA with Dunett’s post hoc analysis (d, e). ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Software, Concentration Assay, Expressing, Western Blot, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) mRNA expression of the FPR family members FPR1, FPR2 and FPR3 at baseline (open bars) or after 2 h of HDM stimulation (filled bars). (b) HDM-triggered IL-33 amounts in BEAS-2B cells overexpressing human FPR1 (**P = 0.0068, ***P = 0.0003). (c) IL-33 secretion in BEAS-2B cells overexpressing human FPR2 or cells transfected with an empty vector (EV; pcDNA3.1) (**P = 0.0068, ***P = 0.0003). HDM-induced IL-6 and IL-8 amounts in BEAS-2B cells overexpressing FPR2 (d and e) or blocking the FPR2 receptor (f (**P = 0.0043) and g (**P = 0.0021)) using WRW4. Data presented as means ± SEM and is representative of 2 independent experiments each containing at least n= 4 biologically independent samples (b, d, f) or pooled data from 2 independent experiments (c, e, g). mRNA expression, normalized to the average of housekeeping genes, is presented as mean values ± SEM (n = 5 biologically independent samples) performed in duplicates (a). P values were calculated with a two-tailed test using Student’s t-test (a) or one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (b-e) or Dunett’s post hoc analysis (f, g). ****P ≤ 0.0001

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing, Transfection, Plasmid Preparation, Blocking Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: IL-33 concentrations in BEAS-2B cells blocking the SAA-binding receptors (a) FPR2 (WRW4, 12 μM; ***P = 0.0006) and (b) TLR4 (LPS from Rhodobacter sphaeroides; LPS-RS, 10 μg/ml; ). (c) Effects of FPR2 or TLR4 blockade in cells transfected with empty plasmid control (EV) or SAA1 overexpression plasmid. **P = 0.0047 § indicates p = 0.056; # indicates p = 0.22 between EV and SAA plasmid. Blockade of (d) CD36 (anti-human CD36 blocking antibody, aCD36; 10 μg/ml; ***P = 0.0005), (e) the P2 receptor antagonist suramin (100 μM; *P = 0.028), and (f) TLR2 (anti-human TLR2 IgA, aTLR2, 10 μg/ml; **P = 0.0055). (g) HDM-triggered IL-33 release in BEAS-2B cells transfected with EV, WT SAA1 overexpression plasmid or a SAA1 plasmid with a deletion of the C-terminal amino acids 1–11 (Δ1–11; *P = 0.01, **P = 0.0078 ). Data are representative of 2–3 independent experiments (b, d-f) or pooled data from 2 independent experiments (a, c, d, g) each containing at least n=4 biologically independent samples and depicted as means ± SEM. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s (a, b, d-g) or Tukey’s (c) post hoc analysis.****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Immunoblot of SAA1 after Alternaria alternata (Alt a) stimulation of BEAS-2B cells performed as described in Fig. 3b. Alt a-induced IL-33 and IL-8 (**P = 0.0044) secretion in BEAS-2B cells after siRNA-mediated silencing of SAA1 (siSAA1) (b and c) or WRW4-mediated FPR2 blockade (d and e). (f) Total serum IgE concentrations, (g) eosinophil counts and frequency of (h) CD3+CD4+, (i) TH2 and (j) TH17 cells in the lungs of PBS or Alt a-treated WT and Saa–/– mice. Immunoblots are representative of an experimental n=2 (a). Data are presented as means ± SEM and represent pooled data from 2 independent experiments (b, d, e, f, g, i, j) or show one representative experiment (c) each containing at least n= 4 biologically independent samples or n=8 WT PBS, n=13 WT Alt a, n=8 Saa–/– PBS and n=11 Saa–/– Alt a animals per group (f, g, i, j) or n=5 WT PBS, n=9 WT Alt a, n=5 Saa–/– PBS and n=7 Saa–/– Alt a animals per group (h). Cropped images are shown. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (a, b) or Dunett’s post hoc analysis (d-j). ****P ≤ 0.0001 siNT = non-targeting siRNA; siSAA1 = SAA1-targeting siRNA. ns=not significant.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Western Blot, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 expression in bronchial epithelial from asthmatic patients and matched controls. Data represents means ± SEM of (a and b) n= 6 control and n= 6 asthmatic patients per group. x.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing