Journal: Frontiers in Cellular and Infection Microbiology

Article Title: PMCNA_RS00975 activates NF-κB and ERK1/2 through TLR2 and contributes to the virulence of Pasteurella multocida

doi: 10.3389/fcimb.2024.1469304

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: Antibodies against TLR2 and TLR4 were purchased from Novus Biological (USA).

Techniques: Sequencing, Mutagenesis

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: PMCNA_RS00975 activates NF-κB and ERK1/2 through TLR2 and contributes to the virulence of Pasteurella multocida

doi: 10.3389/fcimb.2024.1469304

Figure Lengend Snippet: Secretion of IL-6 in response to rPMCNA_RS00975 protein is dependent on TLR2. rPMCNA_RS00975 was pretreated with mouse anti-rPMCNA_RS00975 serum (+anti-rPMCNA_RS00975) at 37°C for 1 h to confirm its neutralizing activity, or pretreated with boiled (+Boiled) at 100℃ for 10 min to confirm that the activation of macrophages was because of rPMCNA_RS00975 and not LPS. (A) TLR4 mRNA levels were determined by qRT-PCR after RAW264.7 macrophages stimulated with the rPMCNA_RS00975 protein (10 μg/mL) for 3 (h) (B) RAW264.7 cells were pretreated for 1 h with anti-TLR2 (5 μg/mL) (+ anti-TLR2), anti-TLR4 (5 μg/mL) (+anti-TLR4) or isotype (+Isotype) before stimulation with the rPMCNA_RS00975 protein (10 μg/mL) for 6 h. The levels of IL-6 in the culture supernatants of RAW264.7 cells were determined by ELISA. (C) Peritoneal macrophages from TLR2 −/− , TLR4, −/− and wild-type mice were incubated with Pam3CSK4 (100 ng/mL), LPS (100 ng/mL), or the rPMCNA_RS00975 protein (10 μg/mL) for 6 h. The levels of IL-6 in the supernatants of cells were measured by ELISA. Data are expressed as means ± SD from three separate experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against TLR2 and TLR4 were purchased from Novus Biological (USA).

Techniques: Activity Assay, Activation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Journal of Traditional and Complementary Medicine

Article Title: Bushen Huoxue Recipe enhances the immune microenvironment at the maternal-fetal interface via the Hippo signaling pathway in mice with recurrent spontaneous abortion

doi: 10.1016/j.jtcme.2025.02.005

Figure Lengend Snippet: Fig. 5. Effect of BSHXR on the expression of inflammatory factors. (A) The mRNA expression of TLR2, TLR4, and TLR7 in decidual tissue (n = 6). (TLR2: Con vs. RSA, p = 0.0036; RSA vs. CsA, p = 0.0443; RSA vs. BSHXR, p = 0.0034; TLR4: Con vs. RSA, p = 0.0097; RSA vs. CsA, p = 0.0260; RSA vs. BSHXR, p = 0.0051; TLR7: Con vs. RSA, p = 0.0104; RSA vs. CsA, p = 0.0315; RSA vs. BSHXR, p = 0.0229). (B) Representative images of immunofluorescence staining for TLR2, TLR4, and TLR7 in decidual tissues. Scale bar: 50 μm. (C) The protein expression of TLR2, TLR4, and TLR7 in decidual tissue. (D) Quantification of the protein levels (n = 3). (TLR2: Con vs. RSA, p = 0.0062; RSA vs. CsA, p = 0.0158; RSA vs. BSHXR, p = 0.0222; TLR4: Con vs. RSA, p = 0.0001; RSA vs. CsA, p = 0.0001; RSA vs. BSHXR, p < 0.0001; TLR7: Con vs. RSA, p = 0.0099; RSA vs. CsA, p = 0.0129; RSA vs. BSHXR, p = 0.0045). The results are expressed as the means ± SEMs. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, ###p < 0.001 and, ###p < 0.0001, compared with the RSA group.

Article Snippet: Antibodies against TLR2 (66645-1-Ig), TLR4 (66350- 1-Ig), TLR7 (17232-1-AP), and GAPDH (60004-1-Ig) were purchased from Proteintech (Rosemont, IL, USA); antibodies against RORγt (ab207082) and FoxP3 (ab215206) were purchased from Abcam (Cambridge, MA, USA); Alexa Fluor 555 nm (8953) and antibodies against MST (14946), p-MST (49332), LATS (3477S), p-LATS (8654S), YAP (14074S), p-YAP (13008S), TAZ (83669S), and p-TAZ (59971S) were purchased from Cell Signaling Technology (Danvers, MA, USA); and an antibody against Lamin B (A11495) was purchased from ABclonal (Wuhan, China).

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: Journal of the American Heart Association

Article Title: Hepatic Abnormal Secretion of Apolipoprotein C3 Promotes Inflammation in Aortic Dissection

doi: 10.1161/jaha.124.037172

Figure Lengend Snippet: Figure 1. Apo C3 is abundant in the plasma of patients with AD and deposits in the aorta. A, Heat map results of plasma exosome sequencing in patients with AD (n=20) and normal controls (n=20). B, ELISA results of apo C3 in the plasma of patients with AD (n=20) and NCs (n=20). C, Representative western blot of apo C3 protein in the aortas of patients with AD (n=6) and NCs (n=6). D, Representative immunohistochemistry of apo C3 in the aortas of patients with AD (n=8) and normal controls (n=8). E, Representative immunofluorescence of apo C3 in the aortas of patients with AD (n=9) and normal controls (n=9). F through H, Quantitative protein levels of apo C3 in western blot (C), immunohistochemistry (D), and immunofluorescence (E). Data are expressed as mean±SEM. Bars represent the means, and caps represent the SEM. Statistical comparisons were made using an unpaired t test (B, F through H). A value of P<0.05 was considered significant. The layers of the aorta are denoted as I for intima, M for media, and A for adventitia. AD indicates aortic dissection; apo C3 apolipoprotein C3; α-SMA, α-smooth muscle actin; CD: cluster of differentiation; ENO, enolase; LPL, lipoprotein lipase; MMP, matrix metalloproteinase; NC, normal control; and RBP, retinol-binding protein.

Article Snippet: Primary antibodies against apo C3 (1:100; Affinity; DF8054), TLR2 (1:100; Proteintech; 17 236- 1- AP), NLRP3 (1:100; Invitrogen; MA5- 23919), CD86 (1:100, Proteintech; 13 395- 1- AP), INOS (1:100; Invitrogen; PA1- 036), MMP2 (1:100; Proteintech; 10 373- 2- AP), MMP9 (1:100; Proteintech; 10 375- 2- AP), CD68 (1:100; Invitrogen; 14- 0688- 82), F4/80 (1:100 Invitrogen; 14–4801- 82), CD31(1:100; Abcam; ab9498) and α- smooth muscle actin (1:100; Boster; BM4172), were incubated overnight at 4°C.

Techniques: Clinical Proteomics, Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry, Immunofluorescence, Dissection, Control, Binding Assay

Journal: Journal of the American Heart Association

Article Title: Hepatic Abnormal Secretion of Apolipoprotein C3 Promotes Inflammation in Aortic Dissection

doi: 10.1161/jaha.124.037172

Figure Lengend Snippet: Figure 3. Transcriptome sequencing reveals inhibition of TLR2/NLRP3 pathway activation, M1 macrophage polarization, and MMP release in the aortas of mice after hepatic apo C3 interference. A through E, Results of transcriptome sequencing in the aortas of mice in the BAPN group (n=5) and BAPN+Sh-apo C3 group (n=5). A, Volcano plot. B, Heat map. C, Protein–protein interaction network of apo C3 with differentially expressed genes. D, GO analysis results. E, KEGG analysis results. apo C3 indicates apolipoprotein C3; BAPN, β-aminopropionitrile; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MMP, matrix metalloproteinase; NLRP3, NOD-like receptor pyrin domain containing 3; and TLR2, Toll-like receptor 2.

Article Snippet: Primary antibodies against apo C3 (1:100; Affinity; DF8054), TLR2 (1:100; Proteintech; 17 236- 1- AP), NLRP3 (1:100; Invitrogen; MA5- 23919), CD86 (1:100, Proteintech; 13 395- 1- AP), INOS (1:100; Invitrogen; PA1- 036), MMP2 (1:100; Proteintech; 10 373- 2- AP), MMP9 (1:100; Proteintech; 10 375- 2- AP), CD68 (1:100; Invitrogen; 14- 0688- 82), F4/80 (1:100 Invitrogen; 14–4801- 82), CD31(1:100; Abcam; ab9498) and α- smooth muscle actin (1:100; Boster; BM4172), were incubated overnight at 4°C.

Techniques: Sequencing, Inhibition, Activation Assay

Journal: Journal of the American Heart Association

Article Title: Hepatic Abnormal Secretion of Apolipoprotein C3 Promotes Inflammation in Aortic Dissection

doi: 10.1161/jaha.124.037172

Figure Lengend Snippet: Figure 1. Apo C3 is abundant in the plasma of patients with AD and deposits in the aorta. A, Heat map results of plasma exosome sequencing in patients with AD (n=20) and normal controls (n=20). B, ELISA results of apo C3 in the plasma of patients with AD (n=20) and NCs (n=20). C, Representative western blot of apo C3 protein in the aortas of patients with AD (n=6) and NCs (n=6). D, Representative immunohistochemistry of apo C3 in the aortas of patients with AD (n=8) and normal controls (n=8). E, Representative immunofluorescence of apo C3 in the aortas of patients with AD (n=9) and normal controls (n=9). F through H, Quantitative protein levels of apo C3 in western blot (C), immunohistochemistry (D), and immunofluorescence (E). Data are expressed as mean±SEM. Bars represent the means, and caps represent the SEM. Statistical comparisons were made using an unpaired t test (B, F through H). A value of P<0.05 was considered significant. The layers of the aorta are denoted as I for intima, M for media, and A for adventitia. AD indicates aortic dissection; apo C3 apolipoprotein C3; α-SMA, α-smooth muscle actin; CD: cluster of differentiation; ENO, enolase; LPL, lipoprotein lipase; MMP, matrix metalloproteinase; NC, normal control; and RBP, retinol-binding protein.

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight at 4 °C with primary antibodies against apo C3 (1:1000; Affinity; DF8054), TLR2 (1:1000; Proteintech; 17 236- 1- AP), NLRP3 (1:1000; Invitrogen; MA5- 23919), cluster of differentiation 86 (CD86) (1:1000; Proteintech; 13 395- 1- AP), inducible nitric oxide synthase (INOS) (1:1000; Invitrogen; PA1- 036), matrix metalloproteinase (MMP) 2 (1:1000; Proteintech; 10 373- 2- AP), and MMP9 (1:1000; Proteintech; 10 375- 2- AP).

Techniques: Clinical Proteomics, Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry, Immunofluorescence, Dissection, Control, Binding Assay

Journal: Journal of the American Heart Association

Article Title: Hepatic Abnormal Secretion of Apolipoprotein C3 Promotes Inflammation in Aortic Dissection

doi: 10.1161/jaha.124.037172

Figure Lengend Snippet: Figure 3. Transcriptome sequencing reveals inhibition of TLR2/NLRP3 pathway activation, M1 macrophage polarization, and MMP release in the aortas of mice after hepatic apo C3 interference. A through E, Results of transcriptome sequencing in the aortas of mice in the BAPN group (n=5) and BAPN+Sh-apo C3 group (n=5). A, Volcano plot. B, Heat map. C, Protein–protein interaction network of apo C3 with differentially expressed genes. D, GO analysis results. E, KEGG analysis results. apo C3 indicates apolipoprotein C3; BAPN, β-aminopropionitrile; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MMP, matrix metalloproteinase; NLRP3, NOD-like receptor pyrin domain containing 3; and TLR2, Toll-like receptor 2.

Article Snippet: The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight at 4 °C with primary antibodies against apo C3 (1:1000; Affinity; DF8054), TLR2 (1:1000; Proteintech; 17 236- 1- AP), NLRP3 (1:1000; Invitrogen; MA5- 23919), cluster of differentiation 86 (CD86) (1:1000; Proteintech; 13 395- 1- AP), inducible nitric oxide synthase (INOS) (1:1000; Invitrogen; PA1- 036), matrix metalloproteinase (MMP) 2 (1:1000; Proteintech; 10 373- 2- AP), and MMP9 (1:1000; Proteintech; 10 375- 2- AP).

Techniques: Sequencing, Inhibition, Activation Assay

Journal: BMC Pulmonary Medicine

Article Title: By activating endothelium histone H4 mediates oleic acid-induced acute respiratory distress syndrome

doi: 10.1186/s12890-024-03334-w

Figure Lengend Snippet: TLR and calcium signaling involved in histone H4-mediated endothelium activation. After the MLVECs were challenged with histone H4 (15 mg/L) for 12 h, the changes in endothelial HS were evaluated by flow cytometry ( A ) while the VE-Cadherin in the cell membrane was measured by western blot ( B ). The vWF in the supernatant ( C, E ) and P-selectin translocation ( D, F ) were measured through ELISA. Prior to histone H4 challenge, the cells were pretreated for one hour with a blocking antibody (10 mg/L) against TLRs (TLR1, TLR2, TLR4, or TLR6) and the calcium chelator EGTA-AM (25, 50 or 100 µM). Data are presented as mean ± SD ( n = 6). The results for flow-cytometry and western blot are representative of three similar samples. * p < 0.05, ** p < 0.01 compared to the control group; # p < 0.05, ## p < 0.01 compared to the H4 group

Article Snippet: The reagents used in this study included: oleic acid (OA) and Histopaque (Sigma-Aldrich St. Louis, MO, USA); histone H4 (Millipore, Billerica, MA, USA); antibodies for P-selectin, sodium-potassium ATPase, CD31 (PECAM-1), and Ly6G (Abcam, Cambridge, MA, USA); an antibody for Cadherin-5 (BD Transduction Laboratories, CA, USA); an antibody for heparan sulfate (HS) (Bioss, Woburn, MA, USA); blocking antibodies against P-selectin, TLR4 (HTA125), TLR2 (TL2.1), and TLR1 (GD2.F4) (eBioscience, San Diego, CA, USA); a blocking antibody against TLR6 (TLR6.127) (Abcam, Cambridge, MA, USA); enzyme-linked immunosorbent assay (ELISA) kits for histone H4 and von Willebrand factor (vWF) (Cusabio Biotech, Wuhan, China); and 1,2-bis(2-aminophenoxy) ethane N, N,N’,N’-tetraacetic acid acetoxymethyl ester (EGTA-AM) (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Activation Assay, Flow Cytometry, Membrane, Western Blot, Translocation Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control