reporter cell lines  (InvivoGen)

Journal: Redox Biology

Article Title: 3- O -trans- p -coumaroyl esterification enhances the anti-inflammatory effects of tormentic acid by targeting NF-κB signaling

doi: 10.1016/j.redox.2025.103731

Figure Lengend Snippet: Effects of TA and trans -TACE on NF-κB activation and oxidative stress-related gene expression in cellular and C. elegans models . (A) NF-κB activation in TLR4-expressing HEK-Blue hTLR4 cells and non-TLR4-expressing HEK-Blue null2 cells following a 24-h treatment with TNFα (10 ng/ml), LPS (10 ng/ml) and TA or trans -TACE (5 μM), normalized to the stress control ( n = 9). (B) Schematic overview of key signaling pathways regulating oxidative stress responses and immune defense in C. elegans . The IIS pathway regulates DAF-16 activity, controlling antioxidant and stress response genes ( sod-3, gpx-5 ). The p38 MAPK pathway, activated via TIR-1, modulates SKN-1-dependent detoxification and immune defense genes ( gcs-1 , gst-4 ). Crosstalk between these pathways ensures coordinated stress adaptation. (C) Relative mRNA expression of daf-16 , sod-3 , gpx-5 , skn-1 , gcs-1 and gst-4 in C. elegans following a 24-h treatment with 50 μM TA or trans -TACE and stress induction by 20 mM PQ, normalized to PQ control ( n = 5 biological, 4 technical replicates). Data are represented as means ± SD of 2–3 independent experiments × p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: The medium was supplemented with 10 % heat-inactivated FBS, 100 U/ml-100 μg/ml P–S and 100 μg/ml NormocinTM (InvivoGen), as well as cell line-specific antibiotics 1X HEK-Blue Selection for the HEK-Blue hTLR4 cell line and 100 μg/ml Zeocin for the HEK-Blue null2 cell line (both from InvivoGen).

Techniques: Activation Assay, Gene Expression, Expressing, Control, Protein-Protein interactions, Activity Assay

Journal: Redox Biology

Article Title: 3- O -trans- p -coumaroyl esterification enhances the anti-inflammatory effects of tormentic acid by targeting NF-κB signaling

doi: 10.1016/j.redox.2025.103731

Figure Lengend Snippet: Comparative effects of trans/ cis -TACE and cis -TACE on key targets of oxidative and inflammatory stress in cellular and C. elegans models. (A) LC-MS chromatograms comparing the cis and trans isoform content of trans -TACE, trans/ cis -TACE and cis -TACE. (B) NF-κB activation in TLR4-expressing HEK-Blue hTLR4 cells and non-TLR4-expressing HEK-Blue null2 cells following a 24-h treatment with TNFα (10 ng/ml), LPS (10 ng/ml) and trans/ cis -TACE or cis -TACE (5 μM), normalized to the stress control ( n = 9–12). (C) Concentrations of TNFα, IL-8, CCL2 and CXCL5 in LPS (250 ng/ml)-stimulated THP-1 cells following a 4-h treatment with 10 μM trans/ cis -TACE or cis -TACE, quantified by multiplex bead-based cytokine immunoassay ( n = 4–6). (D) Intracellular ROS levels in Caco-2 cells after 20 min of treatment with 10 μM trans/ cis -TACE, cis -TACE or quercetin and subsequent stress induction by 300 μM AAPH, normalized to stressor treatment ( n = 12). (E) Relative mRNA expression of daf-16 , gst-4 , gpx-5 , skn-1 , gcs-1 and sod-3 in C. elegans following a 24-h treatment with 50 μM trans/ cis -TACE or cis -TACE and stress induction by 20 mM PQ, normalized to PQ control ( n = 6 biological, 4 technical replicates). Data are represented as means ± SD of 2–3 independent experiments × p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: The medium was supplemented with 10 % heat-inactivated FBS, 100 U/ml-100 μg/ml P–S and 100 μg/ml NormocinTM (InvivoGen), as well as cell line-specific antibiotics 1X HEK-Blue Selection for the HEK-Blue hTLR4 cell line and 100 μg/ml Zeocin for the HEK-Blue null2 cell line (both from InvivoGen).

Techniques: Liquid Chromatography with Mass Spectroscopy, Activation Assay, Expressing, Control, Multiplex Assay

Journal: Infection and Immunity

Article Title: Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization

doi: 10.1128/iai.00369-23

Figure Lengend Snippet: Characterization of purified Lpp20 as model lipoprotein. (A) Lpp20-HA was immunopurified from WT or lnt mutant H. pylori strains VM396 and VM404. A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δlnt Lpp20-HA from strain VM404). (B) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen. Relative amounts of SEAP were measured after 16-h treatment. Results show the mean and standard deviation from single Lpp20-HA preparations (from both WT and lnt mutant H. pylori) tested in triplicate and are representative of three independent preparations of each lipoprotein. (C) Synthetic lipopeptides (0.04 µg/mL triacylated Pam3-CSK4 and diacylated Pam2-CSK4) were used to stimulate reporter cell lines as described in B. Results show the mean and standard deviation of triplicate determinations and are representative of three independent experiments. Results in panels B and C were analyzed by analysis of variance followed by Tukey’s post hoc test. Select pair-wise comparisons are indicated as significantly different (*, P < 0.001) or not significantly different (ns).

Article Snippet: A representative Coomassie-stained gel is shown (lane a, WT Lpp20-HA from strain VM396; lane b, Δ lnt Lpp20-HA from strain VM404). ( B ) Purified Lpp20-HA (0.1 µg/mL from either WT or lnt mutant H. pylori ) was incubated with secreted embryonic alkaline phosphatase (SEAP) reporter cell lines HEK-Blue Null2 (Null), HEK-Blue hTLR4 (TLR4), HEK-Blue hTLR2 KO-TLR1/6 (KO 1/6), HEK-Blue hTLR2-TLR1 (TLR2/1), and HEK-Blue hTLR2-TLR6 (TLR2/6) from InvivoGen.

Techniques: Purification, Mutagenesis, Staining, Incubation, Standard Deviation