Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of 6S on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of 6S on expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1 in HCT-116 cells. (B) Effect of 6S on the expression of Keap1, Nrf2, and phosphorylated Nrf2 (p-Nrf2). The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after treatment of HCT-116 cells with 6S (20 μM). β-Actin was used as an internal standard. (C) Time-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM 6S for 6 h. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E) IF staining of Nrf2. HCT-116 cells were treated with 20 μM 6S for 12 or 24 h and then fixed and labeled with anti-Nrf2 and appropriate FITC-conjugated secondary antibodies. Cells were counterstained with DAPI for visualization of the nuclei. Slides were viewed using fluorescent microscopy (DAPI, blue; Nrf2, red).

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Staining, Labeling, Microscopy

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of M2 on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of M2 on the expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1. (B) Effect of M2 on the expression of Keap1, Nrf2, and p-Nrf2. The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after the treatment of HCT-116 cells with M2 (20 μM). β-Actin was used as internal standard. (C) Time-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM M2 for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM M2 for 6 h. Cytosolic and nucleic Nrf2 were determined using western blotting with the appropriate specific antibodies. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E, F) Effects of kinase inhibitors on M2-induced Nrf2 translocation and phosphorylation (E) and HMOX1 (F) expression. PD098059 (50 μM, a MEK1 inhibitor), LY294002 (50 μM, a PI3K inhibitor), or SB202190 (50 μM, a p38 inhibitor) was used to pretreat the cells for 30 min before they were exposed to 20 μM M2. After another 24 h of incubation, whole-cell lysates were prepared and assessed for Nrf2, p-Nrf2, and HMOX1 expression by western blotting. * p < 0.05.

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Phospho-proteomics, Incubation

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Cysteine Residues of Keap1 Modified by 6S as Determined by UPLC–MS/MS Analysis a

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Modification