il 6 mrna  (Genechem)

Journal: Redox Biology

Article Title: Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

doi: 10.1016/j.redox.2024.103191

Figure Lengend Snippet: Heme is anti-inflammatory in LPS - stimulated human macrophages. hMDMs were treated with various concentrations of heme and with LPS (1 μg/ml) alone or in combination for 16 h, as indicated. (A) Gene expression of TNF-α, IL-6, COX-2 were measured by real-time RT-PCR and cell culture supernatants were collected for ELISA to detect TNF-α and IL-6. (B) Gene expression of HO-1: values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. (C) hMDMs were stimulated with IFN-γ (10 ng/ml), IL-4 (20 ng/ml), LPS or heme for 24 h. Expression of the M1 marker CD64 and the M2 marker CD206 were analyzed by flow cytometry and the fold induction of mean fluorescence intensity relative to unstimulated control macrophages is shown (n = 3). The values represent mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also .

Article Snippet: Primers for quantification of mRNA levels of HO-1, COX-2, IL-6, TNF-α and hypoxanthine phosphoribosyltransferase (HPRT) were from Applied Biosystems.

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Marker, Flow Cytometry, Fluorescence

Journal: Redox Biology

Article Title: Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

doi: 10.1016/j.redox.2024.103191

Figure Lengend Snippet: Heme is pro-inflammatory in LPS stimulated mouse macrophages. mBMDMs were treated with heme (5 or 10 μM) and LPS (1 μg/ml) for 16 h. (A) Gene expression of TNF-α, IL-6, COX-2 were measured by real-time RT-PCR and culture supernatants were collected for ELISA to detect TNF-α and IL-6. (B) Gene expression of HO-1: values were normalized to the expression of HPRT and the respective ΔΔCT values are shown. The values represent mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA with Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Primers for quantification of mRNA levels of HO-1, COX-2, IL-6, TNF-α and hypoxanthine phosphoribosyltransferase (HPRT) were from Applied Biosystems.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: TGF-β-induced interleukin-6 participates in transdifferentiation of human Tenon’s fibroblasts to myofibroblasts

doi:

Figure Lengend Snippet: Multiplex reverse transcription PCR. Multiplex reverse transcription PCR sets for interleukins (ILs; A , B ) and housekeeping genes ( C ). For each set of data, lane 1 represents the control group with no transforming growth factor (TGF)-β1 treatment and lane 2 represents the study group with 10 ng/ml TGF-β1 treatment for 72 h. Lane M contains positive markers. GAPDH , glyceraldehyde-3-phosphate dehydrogenase; IL-1RN , interleukin-1 receptor antagonist; RPL13a ; SDHA , succinate dehydrogenase complex subunit A; β2M , β2-microblobulin. Two asterisks refer to a significant increase (p<0.001) in expression of IL-6 and IL-11 in the presence of TGF-β1.

Article Snippet: siRNA molecules targeting IL-6 and IL-11 mRNA were purchased from Ambion, Inc. (Austin, TX).

Techniques: Multiplex Assay, Expressing

Journal: Molecular Vision

Article Title: TGF-β-induced interleukin-6 participates in transdifferentiation of human Tenon’s fibroblasts to myofibroblasts

doi:

Figure Lengend Snippet: RNA interference assay for IL-6 and IL-11 . After administering small interfering RNAs targeting IL-6 ( A ) and IL-11 ( B ), fibroblasts were exposed to 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. Then, the expression of α-smooth muscle actin (α-SMA) was evaluated using western blots. Densitimetric data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference compared to TGF-β treatment only (p<0.001).

Article Snippet: siRNA molecules targeting IL-6 and IL-11 mRNA were purchased from Ambion, Inc. (Austin, TX).

Techniques: Expressing, Western Blot

Journal: Molecular Vision

Article Title: TGF-β-induced interleukin-6 participates in transdifferentiation of human Tenon’s fibroblasts to myofibroblasts

doi:

Figure Lengend Snippet: Immunofluorescent analysis of the expression of α-smooth muscle actin. A : No treatment control, B : 10 ng/ml transforming growth factor (TGF)-β1 for 72 h, C : IL-6 -specific small interfering (si)RNA, D : IL-6 -specific siRNA plus 10 ng/ml TGF-β1 for 72 h, E : IL-11 -specific siRNA, and F : IL-11 -specific siRNA plus 10 ng/ml TGF-β1 for 72 h. The secondary antibody was conjugated with fluoroscein isothiocyanate, resulting in green fluorescence. Total magnification was 400×.

Article Snippet: siRNA molecules targeting IL-6 and IL-11 mRNA were purchased from Ambion, Inc. (Austin, TX).

Techniques: Expressing, Fluorescence

Journal: Molecular Vision

Article Title: TGF-β-induced interleukin-6 participates in transdifferentiation of human Tenon’s fibroblasts to myofibroblasts

doi:

Figure Lengend Snippet: Promoter deletion assay for interleukin-6 ( IL-6 ). Three recombinant plasmids, in which different fragments of the activator protein-1 (AP-1)-binding site on the human IL-6 promoter were deleted, were used. After transfection with each plasmid, fibroblasts were incubated with 10 ng/ml transforming growth factor (TGF)-β1 for 72 h. The luciferase activity was then determined and expressed as the percent of the no-TGF-β1 treatment control. Data represent the mean±SD of results from three independent experiments. The asterisks refer to a significant difference (p<0.001) compared to the wild type.

Article Snippet: siRNA molecules targeting IL-6 and IL-11 mRNA were purchased from Ambion, Inc. (Austin, TX).

Techniques: DNA Deletion Assay, Recombinant, Binding Assay, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay