human toll like receptor tlr 4 gene  (InvivoGen)

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Primer sequences used for reverse transcription-quantitative PCR analysis.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Sequencing

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds inhibit LPS-induced TLR expression. (A) Western blot analysis of TLR2 and TLR4 expression in THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. (B) Reverse transcription-quantitative PCR analysis of the relative expression levels of TLR2 and TLR4 normalized to GAPDH following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ***P<0.001; # P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Flow cytometry showing the inhibition of LPS-induced TLR4 expression by 48-h treatment with NTS (3 µg/ml), MSM (200 mM) and TLR-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TLR, Toll-like receptor.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Inhibition

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds induce DNA damage response following LPS-induced DNA damage. (A) Images of the comet assay were captured by fluorescence microscopy at ×10 and ×40 magnification levels, showing the fragmented DNA migrating out of the nucleoid body, which formed a comet tail following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. *P<0.05 and ***P<0.001; $ P<0.05 vs. non-treated control; and $$$ P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (B) Western blot analysis of THP-1 cells; NTS (3 µg/ml) and MSM (200 mM) inhibited the LPS-induced expression of p-ATM, p-ATR, p-Chk2, p-BRCA1, and p-p53. However, the expression levels of p-MDM2 were suppressed by LPS treatment, and were increased by NTS (3 µg/ml), MSM (200 mM) or TLR4-C34 (40 µM). LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; p-, phosphorylated; TLR, Toll-like receptor.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Single Cell Gel Electrophoresis, Fluorescence, Microscopy, Control, Western Blot, Expressing

Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Molecular mechanism of LPS-induced regulation of the inflammatory response through iron/heme metabolism and TLR4/NF-κB expression through the canonical NF-κB and PKC-mediated inflammatory pathways. The anti-inflammatory activities of NTS and MSM were achieved by inhibiting iron/heme metabolism and suppressing the expression of TLR4/NF-κB signaling molecules, thus blocking the binding of NF-κB to the gene promoters of proinflammatory cytokines. DMT1, divalent metal transporter 1; Fe 2+ , ferrous ion; FPN, ferroportin; TfR, transferrin receptor; TLR4, Toll-like receptor 4.

Article Snippet: After cultured cells were washed with pre-chilled PBS, cell pellets were incubated with 10% BSA (cat. no. A3311; MilliporeSigma) on ice for 20 min. PE-conjugated anti-human CD284 (TLR4) antibody (1:200; cat. no. 312806; Biolegend, Inc.) and APC-conjugated anti-human CD282 (TLR2) antibody (1:200; cat. no. 309720; Biolegend, Inc.) was used to stain the cells on ice for 30 min. Stained cells were then washed with pre-chilled PBS.

Techniques: Expressing, Blocking Assay, Binding Assay

Journal: bioRxiv

Article Title: Aβ-HMGB1 complex is a pathogenic molecule at the advanced stage of Alzheimer’s disease

doi: 10.1101/2025.06.12.659430

Figure Lengend Snippet: a) Inhibition of the TLR4 – Aβ-dsHMGB1 complex interaction by two types of anti-dsHMGB1 antibody and anti-Aβ antibody was evaluated by ELISA. b) Western blot analysis of MARCKS phosphorylation at Ser46 of normal iPSC-derived neurons before and 30 min after addition of 0.56 nM Aβ-dsHMGB1 complex with or without 0.56 nM lecanemab, anti-dsHMGB1 antibody (CC129) or another anti-dsHMGB1 antibody (CC129L2). Lower graphs show intensities of the band signals corrected with GAPDH. **: p<0.01 in Tukey’s HSD test (N=3 wells per group). c) Immunohitochemistry of normal iPSC-derived neurons after addition of 0.56 nM of Aβ-dsHMGB1 complex with 0.56 nM lecanemab, anti-dsHMGB1 antibody (CC129) or another anti-dsHMGB1 antibody (CC129L2). **: p<0.01 in Tukey’s HSD test (N= 4 wells). d) Live imaging of normal human iPSC-derived neurons cultured during 720 min after addition of 0.56 nM Aβ-dsHMGB1 complex to the medium with one of the three antibodies. Upper panels show representative images of ER ballooning in neurons and lower graphs show the ratio of ER ballooning neurons to total neurons. Mean values of 5 visual fields from 3 wells were quantified. **: p<0.01 in Tukey’s HSD test (N=3 wells). e) The similar live imaging was performed with human iPSC-derived neurons in culture medium added with different concentrations of inhibitors (CC129, CC129L2 and lecanemab). Statistic differences are shown in the suppressive effect of neuronal death evaluated by ER ballooning. *: p<0.05, **: p<0.01, ***: p<0.001 in Tukey’s HSD test (N=3 wells).

Article Snippet: After plates were washed three times with wash buffer (HMGB1 ELISA KIT Exp, 326078738, Shino-Test, Tokyo, Japan), 100 μL peroxidase-labeled (Peroxidase Labeling Kit-NH2, LK11, Dojindo, Kumamoto, Japan) human TLR4 (1478-TR-050, R&D, Minneapolis, MN, USA) at 0, 0.25, 0.5, 1, 2 and 4 □nM in PBS was added to each well and incubated for 24 □hours at 25□°C.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Derivative Assay, Imaging, Cell Culture