Rela p65

Antibodies against SIRT1, CK2α, p53-p21^Cip1/WAF1, RelA/p65, IκB, α-tubulin, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against acetylated RelA/p65 (ac-p65, K310), IKKα/β, phosphorylated IKKα/β (p-IKKα/β, S180/S181), IL-1β, IL-6, MMP3, and histone H3 were obtained from Abcam (Cambridge, UK). Antibodies against phosphorylated IκBα (p-IκBα, S32/S36) and phosphorylated AKT (p-AKT, S473) were obtained from Cell Signaling (Danvers, MA, USA). Resveratrol, triciribine, nicotine amide, and LPS were obtained from the Sigma Chemical Co. (St. Louis, MO, USA).

Total cellular extracts were prepared in lysis buffer (13 mM HEPES, pH 7.9, 350 mM NaCl, 20% glycerol, 1% NP-40, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and protease inhibitors). After centrifugation (15,000× g, 15 min, 4 °C), supernatants were collected. NF-κB double-stranded probe (5′-GAT CCA AGG GAC TTT CCA TG-3′ of the Igk promoter’) was end-labeled with [γ-³²P] ATP using T4 polynucleotide kinase and incubated with samples (10 µg) for 20 min at 30 °C. Cell extracts were preincubated with antibodies (2 µg) specific for p50/NF-κB1, p52, p65/RelA, RelB, c-Rel, or Bcl3 (Santa Cruz Biotechnology) for 30 min on ice before the addition of the labeled probe. DNA/protein and DNA–protein–antibody complexes were resolved on a 5% polyacrylamide gel in 0.5× TBE and detected by autoradiography.

T-84 monolayer: Cells were fixed in methanol and immunostaining was performed using antibodies against occludin (Sigma), E-cadherin (Clone 36; BD Bioscience), desmoglein-2 (Abcam), hemi-desmosome subunit ITGA6 (CD49f, GoH3 monoclonal: BD Bioscience) and p65/RelA (Santa Cruz Biotechnology). For protein visualization, AlexaFluor 488 and 568 antibodies (Invitrogen, Thermo Fisher Scientific) were used. Nuclear staining was performed using DAPI with Vectashield (Vector laboratories) for mounting. Intestinal Organoids: Small intestinal organoids were fixed in 4.5% formaldehyde solution overnight at 37 °C and embedded in Tissue-Tek optimum cutting temperature (O.C.T.) Medium (Sakura Finetek, The Netherlands) and kept at −80 °C. Cryosections were treated with blocking solution followed by primary antibody (E-cadherin; BD Bioscience) incubation (overnight 4 °C) and proteins were visualized using secondary antibody (anti- mouse Alexa Fluor 488; Invitrogen ThermoFisher Scientific). Slides were mounted with Mowiol (Calbiochem). Images were recorded at 400x magnification on an Olympus BX51 microscope, using Cellsens dimension life science imaging software (Olympus; Hamburg, Germany) and processed with Adobe Photoshop (Adobe, Mountain View, CA).

The HeLa, C33A, and Caski cell lines were obtained from American Type Culture Collection (ATCC) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C, 5% CO₂. Anti-MMP2, MMP9, TIMP-1, TIMP-2, β-actin,p38 and p-p38 were obtained from Cell Signaling Technology (Beverly, MA). Anti-NF-κB-p50, p65/Rel A and Histone H1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma (St Louis, MO), unless otherwise indicated.