Ac h3

SRT1720 was supplied by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Doxorubicin (ADR), EX527, wortmannin, insulin, H‐89 and forskolin were supplied by Sigma (St. Louis, MO, USA). Compound C was obtained from Calbiochem (La Jolla, CA, USA). Antibodies were purchased from the following vendors: SIRT1, p‐AMPK (Ser485), H3, p‐AKT, AKT p‐PKA (Thr197) and PKA (Cell Signaling Technology, Danvers, MA, USA); and Ac‐H3, p53, p21, p16, telomerase reverse transcriptase (TERT), SMP‐30 and β‐actin (Santa Cruz Biotechnology).

At the experimental end point, cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 10 min. Following 3 × 5 min washes in 10 mM PBS containing 0.02% Triton X-100 (PBS-T), cultures were incubated in blocking solution (5% BSA in PBS-T) for 1 h at room temperature. Where indicated, cultures were incubated in the following primary antibodies: 11β-HSD2 (1 : 250; Santa Cruz), AcH3 (1 : 250; Santa Cruz), GR (1 : 250; Santa Cruz), or IL1R1 (1 : 250; Invitrogen) diluted in 1% BSA in 10 mM PBS at 4°C for 16 h. Following 3 × 5 min washes in PBS-T, cells were incubated in the appropriate Alexa Fluor 488-conjugated or 594-conjugated secondary antibodies (1 : 1000; Invitrogen) diluted in 1% BSA in 10 mM PBS at room temperature for 2 h. Cultures were counterstained with DAPI (1 : 3000; Sigma). Cells were imaged under an Olympus IX70 inverted microscope with Olympus DP70 camera and AnalysisD™ software.

The LPS, FITC-labeled dextran, calcitriol and ChIP kit were purchased from Sigma Aldrich (St. Louis., MO). The antibodies of VDR, ZO-1, claudin-5, occludin, HDAC11, acH3, acH4, shRNA kits of VDR and HDAC11 were purchased from Santa Cruz Biotech (Santa Cruz, CA). The reagents for Western blotting and RT-qPCR were purchased from Invitrogen (Carlsbad, CA). The reagents used in this study contained <0.2U endotoxin/10 μg reagents as assessed using the Limulus assay (Limulus amebocyte lysate QCL 1000, Bio Whittaker, Walkersville, MD, USA).