Journal: Microorganisms

Article Title: GroEL Secreted from Bacillus subtilis Natto Exerted a Crucial Role for Anti-Inflammatory IL-10 Induction in THP-1 Cells

doi: 10.3390/microorganisms11051281

Figure Lengend Snippet: SDS-PAGE of the affinity-purified GroEL ( A ). Reduction of IL-10-inducing activity of GroEL by the treatments with anti-TLR2 and -TLR4 antibodies ( B ). Significant difference from the control (-), * p < 0.05 (n = 3).

Article Snippet: For receptor analysis, an anti-TLR2 antibody (Sino Biological Inc., Peking, China) or anti-TLR4 antibody (Sino Biological Inc., Beijing, China) was pre-incubated with THP-1 DC prior to the addition of purified GroEL.

Techniques: SDS Page, Affinity Purification, Activity Assay, Control

Journal: Microorganisms

Article Title: GroEL Secreted from Bacillus subtilis Natto Exerted a Crucial Role for Anti-Inflammatory IL-10 Induction in THP-1 Cells

doi: 10.3390/microorganisms11051281

Figure Lengend Snippet: Predicted immunomodulatory effect of GroEL in THP-1 DC. After interactions with a specific receptor, TLR2, STAT, and IRAK2 pathways are activated. Then, transcriptional regulators, JUN, and NF-κB are activated. Finally, specific gene expressions such as cytokines and chemokines are induced.

Article Snippet: For receptor analysis, an anti-TLR2 antibody (Sino Biological Inc., Peking, China) or anti-TLR4 antibody (Sino Biological Inc., Beijing, China) was pre-incubated with THP-1 DC prior to the addition of purified GroEL.

Techniques:

Journal: Redox Biology

Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling

doi: 10.1016/j.redox.2025.103521

Figure Lengend Snippet: Di-HMGB1 binding to TLR4/MD2 and TLR2 . ( A ) Di-HMGB1 was produced by incubating HMGB1 with 10 μM CuCl 2 and 50 μM H 2 O 2 for 18 h at 37°C. ( B ) The TLR4/MD2 complex (1 μg/mL) was coated on a polystyrene microplate for 18 h at 4°C. HMGB1, Di-HMGB1, and HMGB1 C106A proteins were then serially diluted and used for ELISA. BSA was used as a control protein. N = 3. ( C ) The TLR4/MD2 complex (400 ng/mL) was incubated with various HMGB1 proteins (each 100 ng/mL) for 2 h at 37°C. The mixture was then immunoprecipitated with an anti-TLR4 antibody for immunoblotting using anti-HMGB1, anti-MD2, and anti-TLR4 antibodies. The relative band intensities of HMGB1/MD2 were calculated using ImageJ software (NIH). ( D ) His 6 -tagged HMGB1 and (HMGB1) 2 proteins were produced in E. coli . The proteins were observed at the expected size by Coomassie blue staining (left) and Western blotting (right) after SDS-PAGE. ( E – G ) SPR analysis was conducted to study HMGB1 binding to TLR4/MD2. HMGB1 ( E ), (HMGB1) 2 ( F ) and Di-HMGB1 ( G ) were serially diluted starting from 1.4 μM and flowed over TLR4/MD2-immobilized CM5 dextran sensor chip. ( H ) Confocal analysis showing TLR4 oligomerization in RAW264.7 cells. The cells were treated with HMGB1 and (HMGB1) 2 , which had been incubated with 10 μM CuCl 2 and 50 μM H 2 O 2 for 2 h, in the presence or absence of 5 mM DTT. TLR4 was stained with Alexa594-conjugated anti-mouse TLR4 antibody. The fluorescence intensity was calculated using ImageJ software, and the mean fluorescence intensity (MFI) was presented as mean ± SEM (N = 3). ∗p < 0.01, ∗∗p < 0.001, t -test. ( I ) TLR2 (1 μg/mL) was coated on a polystyrene microplate for 18 h at 4°C. Serially diluted HMGB1, Di-HMGB1, and HMGB1 C106A mutant proteins were added and binding was evaluated by ELISA, with BSA as a control protein. N = 3. ( J – L ) SPR analysis of HMGB1 binding to TLR2. HMGB1 ( J ), (HMGB1) 2 ( K ), and Di-HMGB1 ( L ) were flowed over TLR2-immobilized CM5 dextran sensor chip. HMGB1 and (HMGB1) 2 were serially diluted starting from 1.4 μM and flowed over TLR4/MD2-immobilized CM5 dextran sensor chip.

Article Snippet: To compare the binding affinity of HMGB1 with TLR4/MD2 and TLR2 (Sino Biological, 10061-H08B), Di-HMGB1 was prepared.

Techniques: Binding Assay, Produced, Enzyme-linked Immunosorbent Assay, Control, Incubation, Immunoprecipitation, Western Blot, Software, Staining, SDS Page, Fluorescence, Mutagenesis

Journal: Redox Biology

Article Title: ROS anchor PAMPs-mediated extracellular HMGB1 self-association and its dimerization enhances pro-inflammatory signaling

doi: 10.1016/j.redox.2025.103521

Figure Lengend Snippet: A model for PAMPs/H 2 O 2 -mediated HMGB1 homo-dimerization and its pro-inflammatory effects . Passively released extracellular HMGB1, mostly in reduced form, and actively secreted extracellular HMGB1, predominantly in a disulfide form, are initially secreted as monomers. These monomeric forms of HMGB1 can undergo self-association on platforms such as LPS and LTA. The presence of ROS facilitates this process through Cys 106 -Cys 106 -mediated anti-parallel intermolecular dimerization. The resulting Di-HMGB1 exhibits increased binding affinity for TLR4 and TLR2, enhances NF-κB signaling, and elevates TNF-α production compared to monomeric HMGB1.

Article Snippet: To compare the binding affinity of HMGB1 with TLR4/MD2 and TLR2 (Sino Biological, 10061-H08B), Di-HMGB1 was prepared.

Techniques: Binding Assay