Journal: The Journal of Biological Chemistry

Article Title: Epigenetic Regulation of Tumor Necrosis Factor ? (TNF?) Release in Human Macrophages by HIV-1 Single-stranded RNA (ssRNA) Is Dependent on TLR8 Signaling *

doi: 10.1074/jbc.M112.342683

Figure Lengend Snippet: Dependence of TNFα release in human macrophages on TLR8 activation by HIV-1 ssRNA. A and B, intracellular expression of TLR7 and TLR8 in macrophages. THP-1 (A) and AM (B) were incubated with PE-conjugated anti-TLR7 or anti-TLR8 or isotype control antibody, and intracellular expression was determined by flow cytometry. Representative profiles were similar in three independent experiments (n = 3 subjects for AM). C–E, functional silencing of human TLR8 leads to marked diminution of TNFα release in human macrophages. C, flow cytometry analysis of human TLR7/8 after gene silencing with the use of TLR7 or TLR8 siRNA and siRNA or isotype control. A representative flow cytometry tracing shows representative results from one experiment repeated independently in three experiments. N.S., nonsilencing. D an E, THP-1 cells were pretreated with either TLR7 siRNA (D) or TLR8 siRNA (E) or nonsilencing control. Cells were differentiated with phorbol ester, challenged with HIV-1 ssRNA and incubated for 24 h. Cell free supernatant was assayed for TNFα by ELISA. Results are representative of three independent experiments performed in triplicate; *, p < 0.05 when compared with nonsilencing siRNA control. BLP served as positive control. US, unstimulated.

Article Snippet: Anti-TLR7-PE and anti-TLR8-PE antibodies were purchased from Imgenex, San Diego, CA.

Techniques: Activation Assay, Expressing, Incubation, Control, Flow Cytometry, Functional Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Epigenetic Regulation of Tumor Necrosis Factor ? (TNF?) Release in Human Macrophages by HIV-1 Single-stranded RNA (ssRNA) Is Dependent on TLR8 Signaling *

doi: 10.1074/jbc.M112.342683

Figure Lengend Snippet: Dependence of chromatin remodeling at TNFα promoter on HIV-1 ssRNA-induced TLR8 activation. RNAi-based targeted gene silencing of TLR8 in human macrophages was described under ”Experimental Procedures.“ Human macrophage THP-1 cells were pretreated with TLR8 siRNA or nonsilencing control (N.S.). They were then incubated with or without HIV-1 ssRNA for 4 h. A and B, DNA-protein complexes were immunoprecipitated with anti-H3H4me3 antibody (A) or anti-H4Ac antibody (B). Each fraction also included a set of GAPDH primers that served as an internal control. Real-time PCR output data were expressed as a percentage of the input fraction, and statistical analyses were performed. *, p < 0.05 when compared with unstimulated control (US). Data shown are a representative experiment of three independent experiments showing similar results each done in triplicate.

Article Snippet: Anti-TLR7-PE and anti-TLR8-PE antibodies were purchased from Imgenex, San Diego, CA.

Techniques: Activation Assay, Control, Incubation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

doi: 10.3390/ijms16010159

Figure Lengend Snippet: qRT-PCR of Toll-like receptor 1 ( TLR1 ), TLR2 , TLR4 and TLR8 gene expression in normal and cancerous tissue ( * p < 0.05).

Article Snippet: Sections were incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) for 10 min and then followed with anti-TLR1, anti-TLR2, anti-TLR4 or anti-TLR8 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) incubation (1:50 dilution) at room temperature overnight according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

doi: 10.3390/ijms16010159

Figure Lengend Snippet: Immunohistochemistry to analyse protein expression of TLR1, TLR2, TLR4 and TLR8 in normal colon mucosa and cancer tissue obtained from the same patient. The representative pictures show that ( a ) TLR1 immunoreactivity is mainly found in inflammatory cells in normal mucosa (black arrows); ( b ) TLR1 is strongly immunoreactive in cancer cells; ( c ) Some TLR2 immunoreactive cells are present in normal mucosa (black arrow); ( d ) A large number of TLR2 immunoreactive tumor-infiltrating cells are present in cancer tissue (black arrows); ( e ) Some TLR4 immunoreactive cells are present in normal mucosa (black arrow); ( f ) TLR4 immunoreactive cells were not present in cancer tissue; ( g ) TLR8 immunoreactive cells were not present in normal mucosa; and ( h ) Strong TLR8 immunoreactivity is present in the cancer cells.

Article Snippet: Sections were incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) for 10 min and then followed with anti-TLR1, anti-TLR2, anti-TLR4 or anti-TLR8 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) incubation (1:50 dilution) at room temperature overnight according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

doi: 10.3390/ijms16010159

Figure Lengend Snippet: Intensity of TLR1, 2, 4, 8 immunoreactivity.

Article Snippet: Sections were incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) for 10 min and then followed with anti-TLR1, anti-TLR2, anti-TLR4 or anti-TLR8 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) incubation (1:50 dilution) at room temperature overnight according to the manufacturer’s instructions.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

doi: 10.3390/ijms16010159

Figure Lengend Snippet: Clinicopathological features according to TLR1, TLR2, TLR4, TLR8, IL-6, IL-8 and MyD88 expression.

Article Snippet: Sections were incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) for 10 min and then followed with anti-TLR1, anti-TLR2, anti-TLR4 or anti-TLR8 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) incubation (1:50 dilution) at room temperature overnight according to the manufacturer’s instructions.

Techniques: Expressing, Significance Assay

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of TLRs and IL-6 as a Marker in Human Colorectal Cancer

doi: 10.3390/ijms16010159

Figure Lengend Snippet: Primers used in this study. Primer sequences of TLRs and downstream effector molecules including MyD88, IFN-α, IL-6 and IL-8. Primer sequences to amplify β-actin used as a housekeeping control are also indicated.

Article Snippet: Sections were incubated in 2% normal horse serum (Vector, Burlingame, CA, USA) for 10 min and then followed with anti-TLR1, anti-TLR2, anti-TLR4 or anti-TLR8 antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) incubation (1:50 dilution) at room temperature overnight according to the manufacturer’s instructions.

Techniques: Control, Sequencing