Journal: Signal Transduction and Targeted Therapy

Article Title: Eosinophils promote CD8 + T cell memory generation to potentiate anti-bacterial immunity

doi: 10.1038/s41392-024-01752-0

Figure Lengend Snippet: IL-4 inhibits the JNK/caspase-3-mediated CD8 + T cell apoptosis in response to L.m . infection. a – c Representative FACS image and statistical analysis of TLR4-MD2 + ( a ), Annexin-V + ( b ) and PD-1 + ( c ) CD8 + T cells with or without LLO treatment for 4–6 h. d – f Representative FACS image and statistical analysis of Annexin-V + ( d ), PD-1 + ( e ) and cytoplasmic p-JNK + ( f ) of CD8 + T cells with or without JNK inhibitor IQ-1S treatment after L.m . infection. g – i Representative FACS image and statistical analysis of Annexin-V + ( g ), PD-1 + ( h ) and cytoplasmic p-JNK + ( i ) of CD8 + T cells with or without exogenous IL-4 treatment after L.m . infection. j Representative FACS image and quantification of MFI of IL-4R expression in CD8 + T cells with or without treatment of exogenous IL-4. k , l Proportions of Annexin-V + and PD-1 + CD8 + T cells with or without Z-VAD-FMK ( k ) and Z-DEVD-FMK ( l ) treatment after L.m . infection as measured by flow cytometry. m Proportions of cleaved Caspase-3 + CD8 + T cells with or without IL-4 treatment after L.m . infection as measured by flow cytometry. n CFSE analysis of proliferative CD8 + T cells after anti-CD3/CD28 stimulation with IL-4 treatment or not. Data are mean ± SD of one representative experiment. Similar results were seen in two or three independent experiments. Unpaired Student’s t tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Cell Activation Cocktail (with Brefeldin A), Fixation Buffer, APC-Cy7 anti-mouse CD45 (30-F11), PE-Cy7 anti-mouse CD45.1 (A20), PE-Cy7/ Percp-cy5.5 anti-mouse CD4 (RM4-5), APC anti-mouse IFN-γ (XMG1.2), APC anti-mouse PD-1 (29F.1A12), APC/BV605 anti-mouse KLRG1 (2F1), APC anti-mouse CX3CR1 (SA011F11), APC/AF488 anti-mouse CD11b (M1/70), Percp-cy5.5/BV421 anti-mouse CD8a (53-6.7), BV421 anti-mouse Siglec-F (S17007L), purified anti-mouse IL-4 antibody (11B11), PE-Cy7 anti-mouse TLR4-MD2 (MTS510), PE anti-mouse IL-4 (11B11), rmCXCL3, rmCXCL5 were purchased from Biolegend.

Techniques: Infection, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Augmented TLR2 Expression on Monocytes in both Human Kawasaki Disease and a Mouse Model of Coronary Arteritis

doi: 10.1371/journal.pone.0038635

Figure Lengend Snippet: (A) Representative histograms showed surface expression of TLR2 and TLR4 by mean fluorescence intensity (MFI) on peripheral monocytes, neutrophils, and lymphocytes in KD patients before IVIG treatment and control patients. (B) The representative histogram displayed surface expression of TLR2 by MFI on peripheral CD14 + monocytes in human KD patients before and after IVIG treatment as well as those in control patients. (C) TLR2 expression over CD14 + monocytes are expressed as mean±SEM by MFI. n = 6 in controls; n = 12 Pre-IVIG; n = 8 Post-IVIG. ** P <0.01.

Article Snippet: Each staining reaction was incubated for 30 minutes with the following primary antibodies and their corresponding isotypes (all from eBioscience, San Diego, CA) at 4°C as follows: fluorescein isothiocyanate (FITC)-conjugated anti-human CD14, Phycoerythrin (PE)-conjugated anti-human TLR2, PE-conjugated anti-human TLR4, allophycocyanin-labeled anti-mouse CD14, biotin-conjugated anti-mouse TLR2 (subsequently being labeled with streptavidin-peridinin-chlorophyll protein complex), and PE-conjugated anti-mouse TLR4/MD2.

Techniques: Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Augmented TLR2 Expression on Monocytes in both Human Kawasaki Disease and a Mouse Model of Coronary Arteritis

doi: 10.1371/journal.pone.0038635

Figure Lengend Snippet: (A) Representative histograms showed surface expression of TLR2 and TLR4/MD2 by MFI on monocytes, neutrophils, and lymphocytes in the LCWE-treated mice and PBS-treated control mice on post-injection day 7. The numbers labeled over the right upper quadrants indicated the representative percentage of TLR2 + or TLR4/MD2 + cells, corresponding to each cell subpopulation. (B) The surface expression of TLR2 on circulating CD14 + monocytes were analyzed by relative TLR2 MFI (rMFI = TLR2 MFI/isotype MFI) at the indicated time points post-injection. (C) Circulating CD14 + monocytes were significantly increased in LCWE-treated mice on days 3, 7, and 14 post injections. Values are expressed as mean±SME, n = 7 mice/group per time point; * P <0.05; ** P <0.01 versus PBS controls.

Article Snippet: Each staining reaction was incubated for 30 minutes with the following primary antibodies and their corresponding isotypes (all from eBioscience, San Diego, CA) at 4°C as follows: fluorescein isothiocyanate (FITC)-conjugated anti-human CD14, Phycoerythrin (PE)-conjugated anti-human TLR2, PE-conjugated anti-human TLR4, allophycocyanin-labeled anti-mouse CD14, biotin-conjugated anti-mouse TLR2 (subsequently being labeled with streptavidin-peridinin-chlorophyll protein complex), and PE-conjugated anti-mouse TLR4/MD2.

Techniques: Expressing, Injection, Labeling

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: The primer has been used for Realtime-PCR.

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques:

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: Human femoral arteries from patients with angiographic atherosclerotic plaques and interal thoracic artery without plaque were assessed by histological and immunochemical analysis. (A). Sections were stained with hematoxylin and eosin. Or immunofluorescence stains of αSMA and TLR4. (B) Immunohistochemical stains of IL-1β, TNFα, MCP-1 and MMP2; counterstained with hematoxylin. H&E is shown at 40×magnification, and immunofluorescence is shown at 1200× magnification B is shown at 200× magnification. Results are representative of 3 independent experiments. The expression levels of IL-1β, TNF-α, MCP-1 and MMP-2 detected by IHC were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Three different sections and five different fields in each section have been detected. (n = 3, Mean±SD, **P<0.01 compared with control).

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Expressing, Software

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: (A)–(B) Primary SMCs were incubated for increasing amount of time (24, 48 and 96 hrs with 50 ug/mL) and increasing doses (12.5, 25 and 50 ug/mL for 48 hrs) of oxLDL. The expression of TLR4 was detected by Realtime-PCR (A) and Western blotting (B) and quantified by densitometry in 3 independent experiments (A and B) as relative units (TLR4/β-actin). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (C) oxLDL stimulated primary SMCs with increasing doses (12.5, 25 and 50 ug/mL for 24 and 48 hrs). Secretion of IL-1β, TNF-α, MCP-1 and MMP-2 was measured by ELISA and quantified in 3 independent experiments. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with 24 hrs treatment group, ##P<0.01 compared with 12.5 ug/mL oxLDL treatment group, && P<0.01 compared with 25 ug/mL oxLDL).

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques: Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: The primary smooth muscle cells have been cultured from TLR4−/− and C57BL/6 mice. (A) The phenotype of TLR4−/− and C57BL/6 had been detected with Western-blot to assay TLR4 expression. Each type mice choose four different mice. (B) The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and detected the level of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with C57BL/6 mice group).

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques: Cell Culture, Western Blot, Expressing, Incubation

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: The primary smooth muscle cells have been pretreated with TLR4 blocking antibody, with mouse IgG used as control. The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and levels of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium were measured. (Mean ± SD, n = 3; **P<0.01 compared with con group, $$P<0.01 compared with mouse IgG treatment group).

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques: Blocking Assay, Incubation, Cell Culture

Journal: PLoS ONE

Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

doi: 10.1371/journal.pone.0095935

Figure Lengend Snippet: (A)–(C) SMCs were incubated for increasing amount of time (30, 60,120 and 240 min with 50 ug/mL)of oxLDL. The phosphorylation of p38 and NF-κB was detected by Western blotting (A) and quantified by densitometry in 3 independent experiments (B and C) as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (D)- (G) The primary SMCs of TLR4 −/− (C57BL/6 as control) and TLR4 antibody blocking (mouse IgG as control) have been treated with or without oxLDL (50 ug/mL) for 240 min to measure the activation of p38 or NF-κB in 3 independent experiments as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; **P<0.01 compared with oxLDL untreated group, ##P<0.01 compared with C57BL/6 group, $P<0.05, $$P<0.01 compared with mouse IgG group).

Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

Techniques: Incubation, Western Blot, Blocking Assay, Activation Assay