Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Activation of TLR2 is dependent on PPAD expression and activity. U251 MG cells overexpressing the TLR2 receptor were infected (MOI=100) or treated for 4 h with (A) ATCC 33277 strain or Pam3CSK4 (1 μg/ml), n=5-7; (B) various P. gingivalis strains and ATCC-derived isogenic mutants of catalytically inactive PPAD (C351A PPAD), n=3-5; (C) ATCC 33277 wild-type and PPAD mutant strains and sonicates (cells were lysed by sonication (sonic.)), n=4; or (D) outer membrane vesicles (OMVs) isolated from ATCC 33277 strain and its isogenic PPAD mutants (2 µg/ml), n=4. Results in (A) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Results in (B–D) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Data are normalized to those from cells stimulated/infected with the same factor and transfected with an empty vector. Mean + SD; ****p < 0.0001; ***p < 0.001; ns, no statistical significance; 1-way ANOVA both followed by Tukey’s multiple comparisons test. ATCC WT- ATCC 33277.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Expressing, Activity Assay, Infection, Derivative Assay, Mutagenesis, Sonication, Membrane, Isolation, Luciferase, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Fimbriae purified from the wild-type ATCC 33277 strain activate the TLR2 receptor. U251 MG cells overexpressing TLR2 were: (A) infected for 4 h with WT ATCC 33277 and its isogenic major fimbriae (delFimA) mutant strain (MOI=100), n=4-5 (B) infected for 4 h with various ATCC 33277-derived PPAD and FimA mutants as well as the WT W83 strain at different MOI (all strains MOI=20-100 with an additional MOI=5 for WT ATCC 33277), n=3 or (C) infected for 2, 4 or 6 h with WT ATCC 33277 or the PPAD mutant strains (MOI=100), n=3; and (D) treated for 4 h with purified fimbriae (10 µg/ml) from WT ATCC 332771 (FimA WT) or the PPAD mutant (FimA delPPAD) strains; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (E) HEK Blue cells overexpressing TLR2 were treated with purified fimbriae (10 µg/ml) isolated from the WT ATCC 33277 strain (FimA WT) or the PPAD mutant (FimA delPPAD), n=3. Results are presented as the mean ± SD fold induction compared to control (unstimulated) cells. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. In (B) each condition was compared to control uninfected cells and in (C) comparisons were performed for each timepoint separately and significant differences compared to WT ATCC 33277 are depicted in the graph.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Purification, Infection, Mutagenesis, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Isolation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Both active PPAD and fimbriae are crucial for activation of TLR2. U251 MG cells overexpressing the TLR2 receptor were infected for 4 h (MOI=100) with various ATCC 33277-derived isogenic mutants expressing different forms of PPAD (the T1 form, the T2-hyperactive form, and the catalytically inactive C351A mutant form) or FimA; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. ****p < 0.0001; ns, no statistical significance; 1-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Infection, Derivative Assay, Expressing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: The ability of clinical strains to activate TLR2 is much weaker than that of ATCC 33277. (A) Cells were infected for 4 h (MOI=100) with various clinical strains (k1-k10), ATCC 33277 (ATCC WT), or W83, n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (B) Western blot analysis of laboratory and clinical strain cultures (adjusted to OD 600 ) to detect FimA. (C) PPAD activity in whole laboratory and clinical strain cultures (adjusted to OD 600 ), n=6. Results were compared with those from the ATCC 332777 strain. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) The FimA type determined by sequencing of the fimA gene in clinical strains.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Sequencing

Journal: Frontiers in Pharmacology

Article Title: A Series of Genes for Predicting Responses to Anti-Tumor Necrosis Factor α Therapy in Crohn’s Disease

doi: 10.3389/fphar.2022.870796

Figure Lengend Snippet: Experimental validation of core predictors. (A) Coculture system of THP1 and Caco2 cells. (B) Validated Western blotting results for TLR2 overexpression and knockdown. (C) CCK8 results for infliximab and LPS in Caco2 cells. (D) RT-qPCR results for cocultured Caco2 cells after infliximab treatment in the context of LPS-induced inflammation (one-way ANOVA, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were further performed to validate the overexpression and knockdown of TLR2 in THP1 cells (Proteintech, United States).

Techniques: Biomarker Discovery, Western Blot, Over Expression, Knockdown, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Primer/probe information

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques: Immunopeptidomics, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: qPCR confirmation of age-related induction of MHC II pathway component genes

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Gene expression levels of age and cognitive status groups, presented as group mean (% of adult mean) ± SEM

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques: Gene Expression

Journal: Aging (Albany NY)

Article Title: High-mobility group box-1 impedes skeletal muscle regeneration via downregulation of Pax-7 synthesis by increasing miR-342-5p expression

doi: 10.18632/aging.205202

Figure Lengend Snippet: RAGE, TLR2, and TLR4 receptors are involved in the downregulation of Pax-7 expression by HMGB1. C2C12 cells were pre-treated with 100 nM siRNA against RAGE, TLR2, or TLR4 for 24 h and then subjected to HMGB1 (10 ng/mL) stimulation for 24 h. The expression levels of Pax-7 mRNA and protein were respectively analyzed by ( A ) qRT-PCR ( n = 3) and ( B ) western blotting assay ( n = 3). ( C ) Immunofluorescence staining was used to visualize the expression of Pax-7 and desmin in C2C12 cells in DM after HMGB1 treatment (10 ng/mL) with indicated siRNAs ( n = 3). Pax-7 is indicated by red coloring, desmin is indicated by green, and DAPI is shown in blue. Scale bars = 100 μm. ( D ) Number of positively stained cells were quantified by ImageJ software ( n = 3). β-Actin was used as the loading control. All data are presented as the mean ± SD of triplicate experiments. * p < 0.05 compared with control group; # p < 0.05 compared with HMGB1-treated group.

Article Snippet: RAGE (sc-36374), TLR2 (sc-40256), TLR4 (sc-40260), c-Src (sc-29228) siRNAs were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); control siRNA (D-001810-10-05) was purchased from Dharmacon (Lafayette, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Software, Control

Journal: Aging (Albany NY)

Article Title: High-mobility group box-1 impedes skeletal muscle regeneration via downregulation of Pax-7 synthesis by increasing miR-342-5p expression

doi: 10.18632/aging.205202

Figure Lengend Snippet: c-Src signaling is involved in HMGB1-mediated Pax-7 expression and muscle differentiation. Western blot analysis of c-Src phosphorylation in ( A ) C2C12 cells treated with HMGB1 (10 ng/mL) following a time-dependent manner ( n = 3) and ( B ) C2C12 cells pre-treated with various siRNAs against RAGE, TLR2, and TLR4 molecules (100 nM) and then exposed to HMGB1 (10 ng/mL) for 24 h. ( C ) qRT-PCR ( n = 3) and ( D ) western blot analysis of Pax-7 mRNA and protein expression, respectively, in C2C12 cells after HMGB1 co-treatment with c-Src siRNA (100 nM) or c-Src inhibitor (PP2; 1 μM) for 24 h ( n = 3). ( E ) Immunofluorescence staining to visualize the expression of Pax-7 and desmin in C2C12 cells in DM after HMGB1 (10 ng/mL) co-treatment with c-Src siRNA (100 nM) or PP2 (1 μM) for 24 h ( n = 3). Pax-7 is indicated by red coloring, desmin is indicated by green, and DAPI is shown in blue. Scale bars = 100 μm. ( F ) Number of positively stained cells were quantified by ImageJ software ( n = 3). β-Actin was used as the loading control. All data are presented as the mean ± SD of triplicate experiments. * p < 0.05 compared with control group; # p < 0.05 compared with HMGB1-treated group.

Article Snippet: RAGE (sc-36374), TLR2 (sc-40256), TLR4 (sc-40260), c-Src (sc-29228) siRNAs were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); control siRNA (D-001810-10-05) was purchased from Dharmacon (Lafayette, CO, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Immunofluorescence, Staining, Software, Control

Journal: Aging (Albany NY)

Article Title: High-mobility group box-1 impedes skeletal muscle regeneration via downregulation of Pax-7 synthesis by increasing miR-342-5p expression

doi: 10.18632/aging.205202

Figure Lengend Snippet: Graphical abstract: schematic illustration of proposed mechanisms underlying the HMGB1-induced inhibition of muscle regeneration, where HMGB1 inhibits Pax-7 expression and skeletal muscle regeneration by promoting the synthesis of miR-342-5p via RAGE, TLR2, TLR4, and c-Src signaling pathways.

Article Snippet: RAGE (sc-36374), TLR2 (sc-40256), TLR4 (sc-40260), c-Src (sc-29228) siRNAs were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); control siRNA (D-001810-10-05) was purchased from Dharmacon (Lafayette, CO, USA).

Techniques: Inhibition, Expressing, Protein-Protein interactions