n6-ma methyltransferase (mettl3, mettl14, and wtap) antibody sampler kit  (Cell Signaling Technology Inc)

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: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

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 inflammation and iron/heme metabolism. (A) Flow cytometry showing Fe2+ levels in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Western blot analysis of (B) transferrin receptor, ferroportin, DMT1 and STEAP3; and (C) MFRN, ABCB6, ABCB10, ALAS1, HO-1, FECH and FLVCR proteins in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ALAS1, 5′-aminolevulinate synthase 1; DMT1, divalent metal transporter 1; Fe2+, ferrous ion; FECH, ferrochelatase; FPN, ferroportin; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MFRN, mitoferrin; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TfR, transferrin receptor; TLR, Toll-like receptor.

Article Snippet: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

Techniques: Flow Cytometry, Western Blot

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: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

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 inhibit LPS-induced ROS. (A) Western blot analysis of iNOS 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 iNOS mRNA expression in cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Data are normalized to GAPDH. ***P<0.001; # P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (C) Flow cytometric analysis of cellular and mitochondrial ROS in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TLR, Toll-like receptor; ROS, reactive oxygen species.

Article Snippet: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

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

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 the LPS-induced PKC-dependent and canonical NF-κB pathways. Western blot analysis of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the inhibition of LPS-induced (A) p-PKC-α, p-ERK and p-p38 expression; and (B) p-IKKα/β and p-IκBα expression. LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; p-, phosphorylated; TLR, Toll-like receptor.

Article Snippet: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

Techniques: Western Blot, Inhibition, 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: 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: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

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: Sulfur compounds inhibit LPS-induced NF-κB, COX-2 and proinflammatory cytokine expression. (A) Western blot analysis of the expression levels of NF-κB, COX-1 and COX-2 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) RT-qPCR analysis of the relative expression levels of NF-κB, COX-1 and COX-2 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) Western blot analysis of the expression levels of IL-1β, IL-6 and TNF-α 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. (D) RT-qPCR analysis of the relative expression levels of IL-1β, IL-6 and TNF-α normalized to GAPDH in cells 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). (E) Chromatin immunoprecipitation assay of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the relative binding of NF-κB to the promoters of IL-1β, IL-6 and TNF-α. ***P<0.001; # P<0.001 vs. non-treated control (one-way ANOVA and Tukey's test). (F) Nuclear protein extract analysis of THP-1 cells treated with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h showing the protein expression levels of nuclear NF-κB. TBP was used as the housekeeping protein for the nuclear extract and β-actin was used to show the efficacy of nuclear protein extraction. LPS, lipopolysaccharide; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; RT-qPCR, reverse transcription-quantitative PCR; TBP, TATA-binding protein; TLR, Toll-like receptor.

Article Snippet: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Chromatin Immunoprecipitation, Binding Assay, Protein Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction

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: The diluted supernatant was incubated for 5 min at room temperature in wells pre-coated with antibodies (1:50 dilution) specific for IL-1β (cat. no. sc-12742; Santa Cruz Biotechnology, Inc.), TNF-α (cat. no. 6945; Cell Signaling Technology, Inc.) or IL-6 (cat. no. sc-57315; Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. Normal mouse IgG (1:10 dilution; cat. no. M8695; MilliporeSigma) and anti-RNA polymerase II (1:10 dilution; cat. no. R1530; MilliporeSigma) were used as negative and positive controls, respectively.

Techniques: Expressing, Blocking Assay, Binding Assay