md2 g envelope plasmid  (Addgene inc)

Journal: Frontiers in Immunology

Article Title: Carbohydrate fatty acid monosulphate ester adjuvant enhances the immunogenicity of influenza antigens via TLR4/2-dependent mechanisms

doi: 10.3389/fimmu.2026.1787181

Figure Lengend Snippet: CMS activates DCs through TLR4 and TLR2-dependent mechanisms. (A) Heat map representation depicting the expression of different TLRs comparing unstimulated DCs vs CMS-stimulated DCs. (B) THP-1 human monocytic cell line stably expressing a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-kB was treated for 24 hrs with CMS at the indicated concentrations. The activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI−Blue™ Solution. Results of THP1 MD2-CD14-TLR4, THP1 and THP1 KO-TLR4 are shown (mean ± SD, n=4). (C, E) Effect of CLI095 (TLR4 pathway inhibitor), or TL2-C29 (TLR2 pathway inhibitor), compared to DMSO (solvent control) on the expression of CMS-induced DC maturation markers. Scatter plots representing the mean ± SD (n = 8 donors) values of CD80, CD86, and HLA-DR are presented. (D, F) Inhibitory effect of CLI095 or TL2-C29 on the secretion (mean ± SD, n = 8) of IL-8, IL-6, and IL-12p70 (all in pg/ml) by CMS-stimulated DCs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as analyzed by one-way ANOVA with Tukey’s multiple comparisons post-test (C-F) . CA, cells alone; DMSO, dimethyl sulfoxide; SD, standard deviation.

Article Snippet: THP1-Dual, THP1-Dual MD2-CD14-TLR4, and THP1-Dual KO-TLR4 reporter cells (Invivogen) were resuspended at a concentration of 100,000 cells per well in a 96-well U-bottom plate in 200 μl RPMI 1640 Medium, GlutaMAX Supplement (Gibco), supplemented with 25mM HEPES, 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Stable Transfection, Activation Assay, Activity Assay, Solvent, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Carbohydrate fatty acid monosulphate ester adjuvant enhances the immunogenicity of influenza antigens via TLR4/2-dependent mechanisms

doi: 10.3389/fimmu.2026.1787181

Figure Lengend Snippet: CMS activates DCs through TLR4 and TLR2-dependent mechanisms. (A) Heat map representation depicting the expression of different TLRs comparing unstimulated DCs vs CMS-stimulated DCs. (B) THP-1 human monocytic cell line stably expressing a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-kB was treated for 24 hrs with CMS at the indicated concentrations. The activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI−Blue™ Solution. Results of THP1 MD2-CD14-TLR4, THP1 and THP1 KO-TLR4 are shown (mean ± SD, n=4). (C, E) Effect of CLI095 (TLR4 pathway inhibitor), or TL2-C29 (TLR2 pathway inhibitor), compared to DMSO (solvent control) on the expression of CMS-induced DC maturation markers. Scatter plots representing the mean ± SD (n = 8 donors) values of CD80, CD86, and HLA-DR are presented. (D, F) Inhibitory effect of CLI095 or TL2-C29 on the secretion (mean ± SD, n = 8) of IL-8, IL-6, and IL-12p70 (all in pg/ml) by CMS-stimulated DCs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as analyzed by one-way ANOVA with Tukey’s multiple comparisons post-test (C-F) . CA, cells alone; DMSO, dimethyl sulfoxide; SD, standard deviation.

Article Snippet: THP1-Dual, THP1-Dual MD2-CD14-TLR4, and THP1-Dual KO-TLR4 reporter cells (Invivogen) were resuspended at a concentration of 100,000 cells per well in a 96-well U-bottom plate in 200 μl RPMI 1640 Medium, GlutaMAX Supplement (Gibco), supplemented with 25mM HEPES, 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Expressing, Stable Transfection, Activation Assay, Activity Assay, Solvent, Control, Standard Deviation

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Interaction of GA monomers with the MD2/TLR4 complex. (A–H) Surface plasmon resonance (SPR) analysis showing direct binding of Ganoderic acid A, B, C2, C6, G, H, K, and Ganoderenic acid B to MD2. (I) Binding of GA‐A to MD2 as assessed by protein microarray analysis. (J) Identification of MD2/TLR4 complexes via immunoprecipitation.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: SPR Assay, Binding Assay, Microarray, Immunoprecipitation

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Molecular docking of GA‐K with MD2 and its effect on cerebral ischemic injury in the mouse tMCAO model. (A) Molecular docking of GA‐K (yellow) with the MD2 protein (green), analyzed using the Trips molecular modeling software. (B) Representative coronal brain sections stained with TTC, showing typical infarct areas in white. Scale bar = 5 mm. (C) Quantification of infarct volume. (D) Neurological deficit scores quantification. The data are presented as the mean ± SEM ( n = 8). Statistical significance: ** p < 0.01 compared to the tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Software, Staining

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: GAs suppress MD2/TLR4 complex formation and inhibit MAPK and NF‐κB signaling pathways in a mouse model of tMCAO. GA (administered at doses of 0 or 20 mg kg −1 , i.p.) was given immediately after reperfusion. At 24 h post‐reperfusion, total and nuclear proteins were isolated from the cortical penumbra for analysis by Western blotting. (A) Immunoprecipitation analysis of the MD2/TLR4 complex in the ischemic hemisphere. (B) Quantification of MD2 expression levels. (C) Representative Western blot images showing proteins involved in the MAPK signaling pathway. (D) Quantitative analysis of phosphorylation levels. (E) Representative Western blot images of nuclear NF‐κB and AP‐1. (F) Quantification of protein expression. The data are presented as the mean ± SEM ( n = 4). Statistical significance is indicated as follows: ### p < 0.001 compared to the sham group, * p < 0.05, ** p < 0.01 compared to the vehicle‐treated tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Protein-Protein interactions, Isolation, Western Blot, Immunoprecipitation, Expressing, Phospho-proteomics

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: MD2 knockout reduces microglia activation and improves acute cerebral ischemic injury in the tMCAO mouse model. (A) Representative micrographs (magnification ×100) showing immunofluorescent staining of MD2 (red) in the peri‐infarct area of the cortex and the dentate gyrus of the hippocampus, 24 h after reperfusion. Scale bars: 50 µm. WT and MD2‐KO mice underwent 1 h of tMCAO, followed by 24 h of reperfusion. GA (0 or 20 mg kg −1 , i.p.) was administered immediately post‐reperfusion. (B) Representative micrographs depicting immunofluorescence for Iba‐1 (green). Primary microglial cells were isolated from WT and MD2‐KO mice, pretreated with GA (50 µg mL −1 ) or vehicle for 1 h, then stimulated with LPS (10 ng mL −1 ) for 12 h. (C) Representative Western blots illustrating levels of p‐JNK, p‐ERK, p‐P38, and p‐NF‐κB. (D) Representative Western blots for inflammatory mediators iNOS, COX‐2, and TNF‐α ( n = 4). (E) Representative coronal brain sections stained with TTC. Infarct areas appear white. Bar = 5 mm. (F) Infarction volume assessment. (G) Neurological deficit score quantification. The data are presented as the mean ± SEM ( n = 8). Statistical significance is indicated as follows: * * P < 0.01, ** * P < 0.001 compared to the WT tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Knock-Out, Activation Assay, Staining, Immunofluorescence, Isolation, Western Blot

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Proposed mechanism of GA in alleviating cerebral ischemic injury. GA monomers interact directly with MD2, preventing the dimerization of MD2 and TLR4, as well as the subsequent activation of downstream MAPK and NF‐κB signaling pathways. This process lowers inflammatory mediator production and reduces microglial overactivation.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Activation Assay, Protein-Protein interactions

Journal: Oncoscience

Article Title: Targeting MD2 in prostate cancer bone metastasis: Mechanistic insights and therapeutic potential

doi: 10.18632/oncoscience.647

Figure Lengend Snippet: ( A ) Detection of HMGB1 in CM from a panel of prostate cancer (CaP) cell lines, including LNCaP control (PCMV vector) and MD2-overexpressing cells (M2 vector). ( B ) HMGB1 levels in CM from DU145 control cells (SCR-siRNA) and MD2-silenced cells (MD2 siRNA). ( C ) Levels of soluble MD2 (sMD2) and HMGB1 in CM from LNCaP cells overexpressing MD2 after treatment with the MD2 inhibitor (MD2-int-1) or vehicle control for 24 h.

Article Snippet: Once tumors were established, as confirmed by IVIS bioluminescence imaging, mice were randomized to receive either vehicle or the selective small molecule MD2 inhibitor, MD2-int-1 (MedChemExpress), for three weeks.

Techniques: Control, Plasmid Preparation