Ad cre

Macrophages were developed from bone marrow cells flushed from the femurs of three wild-type or SIRT1^f/f mice (Jackson Laboratories) cultured in α-MEM supplemented with 10% FBS and 1% PSG (Invitrogen) in the presence of 30 ng/ml M-CSF (R&D Systems, Minneapolis, MN). Four days later cells were infected with adenovirus encoding Cre recombinase (Ad-Cre) (Vector Biolabs, Philadelphia, PA) at a MOI of 30 for 6 hours. Seventy-two hours later cells were trypsinized and re-plated in 48-well plates and cultured for 4 days with 30 ng/ml M-CSF and 30 ng/ml RANKL (R&D Systems) to obtain osteoclasts, in the presence of vehicle or SRT2104. At the end of the experiment, osteoclasts were fixed with 10% neutral buffered formalin for 15 minutes and stained for tartrate-resistant acid phosphatase (TRAP). Multinuclear TRAP+ cells were quantified.

Adenoviruses for red fluorescent protein (Ad-RFP) and Cre recombinase (Ad-Cre) (Vector Biolabs, Malvern, PA, USA) were applied at a multiplicity of infection (MOI) 0, 10, 100 and, 200. After three days, TPCs were fixed in 4% paraformaldehyde and stained with DAPI. RFP+ or RosaT+ cells and DAPI+ nuclei were quantified in 3 representative images per well, and the number of RFP+/DAPI+ and RosaT+/DAPI+ cells was determined.

The following adenoviruses were purchased: Ad-GFP-m-SIK1 (ADV-271991, Vector Biolabs) and Ad-CMV-GFP (1060, Vector Biolabs) as a control. Dissociated mouse islets were transduced with adenoviruses for 2 h in RPMI 1640 medium (21875034, Thermo Fisher Scientific) without FBS at an MOI of 50. The wells were then washed, and RPMI 1640 (21875034, Thermo Fisher Scientific) containing 10% FBS and 15 mM glucose was added to the wells. After 4 h, cells were treated with HG (0.1, 0.05 or 0.025 μM) or with DMSO (1%) in the presence of EdU (10 μM). After 4 d, cells were either fixed for immunofluorescence studies or lysed to assess mRNA expression. Sik1 and Hprt (control) primer sequences are shown in Supplementary Table 8. For experiments with MIN6, cells were transduced with adenoviruses for 4 h in DMEM-based medium (C0003–04, AddexBio) without FBS at an MOI of 50. The wells were then washed, and DMEM-based medium (C0003–04, Addexbio) containing 15% FBS and 0.055 mM β-mercaptoethanol was added to the wells. After 4 d, cells were treated with HG (1 μM) or DMSO (1%) for 6 h. For the experiments presented in Fig. 7, islet cells from Atf6 floxed mice were transduced with Ad-LacZ (1080, Vector Biolabs) or Ad-Cre (1045, Vector Biolabs) at an MOI of 10 for 72 h in RPMI 1640 medium containing 10% FBS and 15 mM glucose.