Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: Immunohistochemistry showing ( A ) TLR7 was highly expressed on cell membrane and perinuclei only in malignant hepatocytes but as the scattered spots in cytoplasm of hepatocytes of Normal, CHB and LC. ( B ) Correlation of TLR7 and Ki-67 expression in HCC. Serial sections from the same paraffin block were stained with different antibodies. Hepatic expression of the proliferative marker Ki-67 was high in nuclei. Membranous TLR7 expression was also high in malignant hepatocytes. ( C ) Percentage of cases from the validation set demonstrating no (0), weak (1) or high (2) expression of TLR7. ( D ) Correlation of TLR7 staining with Ki-67 index in HCC. ( E ) Western blotting analysis of TLR7 in liver tissue lysates. Bands were observed at approximately 120 kDa and normalized by β-actin. There was a significant increase in the TLR7 expression of HCC group compared with Normal control. * P < 0.05, results were representative of three independent experiments.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Membrane, Expressing, Blocking Assay, Staining, Marker, Biomarker Discovery, Western Blot, Control

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: Tissue clinical data and TLR7 staining

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Membrane

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: To evaluate the efficacy of the experiment, an isotype-matched control was used. At least 3000 cells were assessed to calculate the mean fluorescence intensity values (MFI). ( A ) Statistical analyses of TLR7 MFI. Data were shown as mean ± SEM. * P < 0.05 compared with the Normal group. ( B ) Expression of TLR7 using flow cytometry analysis. ( C ) The immune cells were identified and evaluated by flow cytometry for the further observation on TLR7-related immune cell infiltrates. NK cells and pDC were in association to TLR7 expression levels. CD3, for T cells; CD8, for cytotoxic T cells; CD19, for B cells; CD56, for NK cells or NKT cells; CD16, for granulocytes; CD14, for mononuclear phagocytes; CD11c, for pDC.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Control, Fluorescence, Expressing, Flow Cytometry

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: ( A ) Lipid raft and non-raft microdomains were isolated using a non-detergent method and analyzed for caveolin-1 (lipid raft marker), flotillin-1 (lipid raft marker), clathrin (non-raft marker) and TLR7 distribution by western blotting. The graph showed the expression of caveolin-1, flotillin-1, clathrin and TLR7 in HCC relative to Normal tissue. * P < 0.05, lipid rafts compared with the non-rafts. ( B ) The co-expression of TLR7 and lipid rafts was analyzed by double immunohistochemistry. The co-expression of TLR7 and lipid rafts was located in the cell membrane only in HCC group but as the scattered spots in cytoplasm of live cells in Normal, CHB, LC groups. Interestingly, margination orientation of TLR7/Caveolin-1 or TLR7/Flotillin-1 from cytoplasm to membrane was observed in LC tissues. The microphotographs were magnified 1000 times.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Isolation, Marker, Western Blot, Expressing, Immunohistochemistry, Membrane

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: ( A ) HepG2 cells were incubated with 10 mM MβCD or 1 μg/ml gardiquimod for 1 h. Cell lysates were harvested and the proteins were analyzed by immunoblotting with the indicated antibodies. ( B ) TLR7, MyD88, NFκB were quantified and normalized to β-actin. Data were presented as mean ± SEM; n = 3. Statistically significant differences between groups were indicated (* P < 0.05). ( C ) HepG2 cells were treated with 10 mM MβCD or 1 μg/ml gardiquimod for 1h and then lipid rafts (fractions 4–6) were pooled, and immunoprecipitated with anti-flotillin-1 polyclonal antibody, followed by immunoblotting with anti-TLR7 monoclonal antibody. Results were representative of three independent experiments. ( D ) Lipid rafts isolated as described in panel C were immunoprecipitated with anti-TLR7 monoclonal antibody, followed by immunoblotting with anti-flotillin-1 polyclonal antibody. Results were representative of three independent experiments.

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Incubation, Western Blot, Immunoprecipitation, Isolation

Journal: Oncotarget

Article Title: Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

doi: 10.18632/oncotarget.11697

Figure Lengend Snippet: TLR7 immunohistochemistry scoring system

Article Snippet: Mouse IgG 2A anti-Human TLR7 PE-conjugated monoclonal antibody and mouse IgG 2A PE-conjugated isotype control were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Staining

Journal: Journal of Innate Immunity

Article Title: TLRs1-10 Protein Expression in Circulating Human White Blood Cells during Bacterial and COVID-19 Infections

doi: 10.1159/000536593

Figure Lengend Snippet: Box plot representation of intracellular expression of TLR3 and TLR7 in neutrophils in septic shock and COVID-19 patients compared to healthy donors. The results are presented as mean fluorescence intensity (MFI). The main body of the boxplots show the 1st and 3rd quartiles. Horizontal lines in each box represent the median. * p < 0.05, ** p < 0.005.

Article Snippet: Within 3 h, blood cells were stained using 14 monoclonal antibodies coupled to a fluorescent dye for the 10 TLRs, CD14, CD16, and CD19 antigens: CD19-APC (clone SJ25C1, #17-0198-42); TLR1-PE (clone GD2.F4, #12-9911-42); TLR3-PE (clone TLR3.7, #12-9039-82), TLR10-PE (clone 3C10C5, #12-2909-42) from Invitrogen; CD16-APC-H7 (clone 3G8, #560195); CD14-PE-Cy7 (clone Mφ9, #562698); TLR4-BV421 (clone TF901, #564401); CD180-BV421 (clone G28-8, #743624) from BD Biosciences; TLR2-PerCP (clone 383936, #FAB2616C); TLR5-A488(clone 624915, #FAB6704G); TLR7-PerCP (clone 533707, #IC5875C); TLR8-A488 (clone 935166, #IC8999G); TLR9-A405 (clone 26C593R, #IC36583V) from R&D Systems and TLR6- FITC (clone TLR6.127, #ab72362) from Abcam.

Techniques: Expressing, Fluorescence

Journal: Scientific reports

Article Title: COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

doi: 10.1038/s41598-022-26457-8

Figure Lengend Snippet: Figure 6. Cytokine production and TLRs in response to plasma exosomes. (a) PBMCs were treated with the TLR3 inhibitor (10 µM) for 30 min or remained untreated, followed by stimulation with plasma exosomes (4 × 109 ml−1) for 16 h. Production of cytokines was determined by flow cytometry gated on CD4+, CD8+, and CD14+. COV exo, COVID-19 plasma exosome treatment; inh/cov, TLR3 inhibitor treated and COVID-19 plasma exosomes stimulated; non-COV, treatment with non-COVID plasma exosomes. *p < 0.05; ns, p > 0.05. ANOVA, equal variants. (b) CD4+ T cells, CD8+ T cells, and CD14+ monocytes were isolated from PBMC using MicroBeads, followed by treatment with plasma exosomes from COVID-19 patients upon admission (COV exo, 4 × 109 ml−1), non-COVID donors (non- COV exo, 4 × 109 ml−1), or poly(I:C) (5 µg ml−1) for 16 h at 37 °C. Expression of TLR3, TLR7, TLR8, and TLR9 was determined by flow cytometry gated on live cells. Isotype antibodies and no-antibody blanks were used in each flow cytometry assay. ctrl, medium only control. Error bars, ± SD; *p < 0.05; ns, p > 0.05; one-way ANOVA. Isotype antibody controls and blank controls were performed in parallel in flow cytometry.

Article Snippet: For flow cytometry, cells were stained using monoclonal antibodies: PE-conjugated IL-6 (Clone MQ2-13A5, BD Biosciences, Franklin Lakes, NJ), APC-conjugated TNF-α (Clone MAb11, BD Biosciences), PE-CF594-conjugated IL-8 (Clone G265-8, BD Biosciences), PE-CF594-conjugated IL-10 (Clone JES3-19F1, BD Biosciences), APC-R700-conjugated IL-17 (Clone N49-653, BD Biosciences), PerCP-Cy5.5-conjugated IFNγ (Clone B27, BD Biosciences), PerCP-Cy5.5-conjugated TGFβ (Clone TW4-9E7, BD Biosciences), PE-conjugated TLR3 (Clone TLR-104, Biolegend, San Diego, CA), PerCPconjugated TLR7 (Clone 533707, R&D systems), FITC anti-human CD288 (TLR8) antibody (Clone 16018A, Biolegend), and APC-conjugated TLR9 (Clone S16D, Biolegend).

Techniques: Clinical Proteomics, Flow Cytometry, Isolation, Expressing, Control