Journal: Nutrients

Article Title: Remission Effects of Dietary Soybean Isoflavones on DSS-Induced Murine Colitis and an LPS-Activated Macrophage Cell Line

doi: 10.3390/nu11081746

Figure Lengend Snippet: Effects of SIF on NF-κB transactivation and LPS-induced IL-6 reporter luciferase activity. ( A ) RAW264.7 macrophages were transfected with the indicated mIL-6-NF-κB-WT-Luc or mIL6-NF-κB-Mut-Luc plasmid together with pRL-TK Renilla luciferase plasmid (pRL-TK). After 48 h, the cells were deprived of serum for 12 h, then lysates were prepared, and luciferase activity was evaluated 4 h after LPS treatment. Data represent the mean ± S.D. (n = 4, significant compared with each non-treated group, *** p < 0.001, ns indicates not significant) ( B ) RAW264.7 macrophages were transfected with mIL6-NF-κB-WT-Luc and pRL-TK Renilla luciferase plasmids (pRL-TK), and 48 h later the cells were pre-treated with SIF for 12 h and LPS (0.3 µg/mL) was added for 4 h. Data represent the mean ± S.D. (n = 8, significant compared with LPS alone, * p < 0.05, **** p < 0.0001, ns indicates not significant).

Article Snippet: Briefly, the cells were transiently co-transfected with 200 ng of mIL6-NF-κB-WT-Luc or mIL6-NF-κB-Mut-Luc and 20 ng of the pRL-TK plasmid (Renilla luciferase expression for normalization) (Promega) using X-treme Gene 9 (Roche).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Nutrients

Article Title: Remission Effects of Dietary Soybean Isoflavones on DSS-Induced Murine Colitis and an LPS-Activated Macrophage Cell Line

doi: 10.3390/nu11081746

Figure Lengend Snippet: Effects of SIF treatment on LPS-induced NF-κB nuclear translocation via the IKK/IκB signaling pathway. ( A ) Confocal microscope images of RAW264.7 cells stained with anti-NF-κB-p65 antibody (Alexa 488, green) and nuclei (DAPI, blue). RAW264.7 cells were pre-treated with SIF (1 mg/mL) for 12 h prior to LPS treatment for 15 min. Microscopic images of p65, nuclei and merged signals (Alexa 488 and DAPI) are shown in the upper, middle, and lower panels, respectively. All images were taken using an LSM-780 microscope (Carl Zeiss). Original magnification: ×63 with ×3 digital zoom, oil objective. Scale bar for the images indicates 5 µm. ( B ) IKK phosphorylation was detected using an immunoblotting assay with anti-phospho-IKKα/β, total-IKKα and IKKβ monoclonal antibodies, respectively. ( C ) Phosphorylation of p65 was detected using phospho-NF-κB-p65 and NF-κB-p65 antibodies. ( D ) IκB degradation was detected using an anti-IκBα monoclonal antibody. RAW264.7 cells were pre-treated with SIF at different concentrations (0–1 mg/mL) for 12 h. Total cell lysates were collected after 15 min LPS (0.3 µg/mL) treatment. GAPDH was used as a loading control. ( E ) Densitometric quantification of IKK phosphorylation, phosphorylation of p65 and IκB degradation were shown in the left, middle and right panels, respectively. The graph calculated by averaging the results were normalized each internal control from three independent experiments. Data represent the mean ± S.D (ratio to Con, significant compared with LPS alone, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Briefly, the cells were transiently co-transfected with 200 ng of mIL6-NF-κB-WT-Luc or mIL6-NF-κB-Mut-Luc and 20 ng of the pRL-TK plasmid (Renilla luciferase expression for normalization) (Promega) using X-treme Gene 9 (Roche).

Techniques: Translocation Assay, Microscopy, Staining, Western Blot