Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA-129/FBW7/NF-κB, a Novel Regulatory Pathway in Inflammatory Bowel Disease

doi: 10.1016/j.omtn.2019.10.048

Figure Lengend Snippet: FBW7 Decreases IκBα Expression and Promotes NF-κB Activation (A) Caco-2 cells were transfected with FBW7 siRNA (20 nM) or negative siRNA (negative) for 48 h. FBW7 mRNA expression was determined by RT-PCR. (B) The cells were infected with adenovirus encoding FBW7-GFP (FBW7-GFP, 50 MOI) or GFP for 48 h. RT-PCR analysis of FBW7 mRNA expression. **p < 0.01 versus control, n = 6. (C and D) Western blotting analysis of FBW7, IκBα, and p65 expression and IκBα phosphorylation in cells treated with FBW7 siRNA (C) or FBW7-GFP adenovirus (D). FBW7 knockdown significantly increased IκBα expression and decreased IκBα phosphorylation and p65 expression, whereas FBW7 upregulation reduced IκBα expression and increased IκBα phosphorylation and p65 expression. Representative images from six independent repetitions were shown. (E–H) The mRNA expression of TNF-α (E), IL-1β (F), IL-6 (G), and IL-8 (H) was examined by RT-PCR. **p < 0.01 versus negative siRNA or GFP adenovirus, n = 6. (I and J) Caco-2 cells were treated with FBW7 siRNA (I) and FBW7-GFP adenovirus (J) for 48 h, and then cycloheximide (CHX) was added at 10 μg/mL for the indicated times. IκBα expression was determined by western blotting. The degradation of IκBα-induced cycloheximide was significantly inhibited by FBW7 downregulation but enhanced by FBW7 overexpression; n = 4. (K and L) Western blotting analysis of IκBα expression in Caco-2 cells pretreated with MG132 (10 μg/mL) (K) or chloroquine (10 μg/mL) (L) for 30 min, followed by FBW7 siRNA for another 48 h. MG132, but not chloroquine, significantly reversed the inhibitory effect of FBW7 knockdown on IκBα expression. n = 5. (M) Cell lysates were immunoprecipitated with IκBα antibody, and immunoprecipitated proteins were blotted with FBW7 antibody; n = 4. (N) Caco-2 cells were cotransfected with HA-IκBα plasmid and FBW7-GFP adenovirus. HA-IκBα was immunoprecipitated (IP) and immunoblotted (IB) with GFP antibody; n = 4. (O and P) Cell were cotransfected with myc-ubiquitin (myc-Ub), HA-IκBα, and FBW7 siRNA (O) or FBW7-GFP adenovirus (P). After immunoprecipitation with HA antibody, proteins were immunoblotted with myc antibody to show IκBα ubiquitination; n = 6.

Article Snippet: Adenovirus encoding human FBW7 cDNA and GFP were provided by Sunbio Biotechnology (Shanghai, China).

Techniques: Expressing, Activation Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Control, Western Blot, Phospho-proteomics, Knockdown, Over Expression, Immunoprecipitation, Plasmid Preparation, Ubiquitin Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA-129/FBW7/NF-κB, a Novel Regulatory Pathway in Inflammatory Bowel Disease

doi: 10.1016/j.omtn.2019.10.048

Figure Lengend Snippet: miR-129 Suppresses IκBα Ubiquitination and Increases IκBα Expression (A) Caco-2 cells were transfected with miR-129 mimics (MiR-129-m) or mimics negative control (NC-m) in the presence or absence of FBW7-GFP adenovirus treatment for 48 h. IκBα protein expression was examined by western blotting. (B) miR-129 inhibitor (MiR-129-i; 10 nM) or inhibitor negative control (NC-i) was cotransfected with or without FBW7 siRNA into cells. IκBα protein expression was determined. **p < 0.01 versus mimics negative control or inhibitor negative control; ##p < 0.01 versus miR-129 mimics or inhibitor; n = 6. (C and D) Caco-2 cells transfected with myc-ubiquitin (myc-Ub) and HA-IκBα were treated as above. HA-IκBα was immunoprecipitated (IP) and immunoblotted (IB) with myc antibody. Upregulation of FBW7 reversed the miR-129-induced decrease in ubiquitination of IκBα (C), while miR-129 inhibitor failed to induce ubiquitination of IκBα in FBW7 siRNA-treated cells (D); n = 5.

Article Snippet: Adenovirus encoding human FBW7 cDNA and GFP were provided by Sunbio Biotechnology (Shanghai, China).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Negative Control, Western Blot, Immunoprecipitation