Rats, Inbred F344

We sequenced the genomes of 25 rat strains, namely ACI/EurMcwi, BBDP/Wor, F344/NCrl, FHH/EurMcwi, FHL/EurMcwi, GK/Ox, LE/Stm, LEW/Crl, LEW/NCrlBR, LH/MavRrrc, LN/MavRrrc, LL/MavRrrc, MHS/Gib, MNS/Gib, SBH/Ygl, SBN/Ygl, SHR/NHsd, SHRSP/Gla, SS/Jr, SS/JrHsdMcwi, SR/Jr, WAG/Rij, WKY/Gla, WKY/NCrl, WKY/NHsd on the Illumina sequencing platform (Table 1). We also included in our analysis the genomes of two rat strains SHR/OlaIpcv and BN.LX that we had sequenced previously on the Illumina and SOLiD sequencing platform respectively (Atanur et al., 2010; Simonis et al., 2012 ), taking the number of analyzed genomes to 27.

A Cre-inducible SIRT1 expression construct was generated in which a loxP flanked transcriptional STOP element was inserted between a CAGGS promoter and the SIRT1 cDNA. This construct was targeted into the mouse Collagen A1 locus using flp recombinase-mediated genomic integration as described previously (1). MES cells carrying a single copy of the SIRT1^STOP construct were identified by resistance to the antibiotic marker hygromycin and Southern blotting. PCR primers and construct maps are available upon request. Two clones were injected into blastocysts and both generated pups, ∼90% of which displayed germ-line transmission. Tumor bearing mice that were analyzed had been backcrossed at least four generations into C57/BL6. APC^min/+ and Villin-Cre transgenic mice strains were obtained in the C57/BL6 background from Jackson Labs (Bar Harbor, ME). SirT1^STOP animals were backcrossed two generations into C57BL/6 mice before crossing to APC^min/+ to generate SirT1^STOP; APC^min/+ double transgenics. These animals were bred to Villin-Cre transgenic mice to generate a cohort of SirT1^ΔSTOP; Vil-Cre; APC^min/+ animals. Animals were maintained at Harvard Medical School and experiments were approved by the Animal Care Committee of Harvard Medical School.
Male Fischer-344 (F344) rats were bred and reared in a vivarium at the Gerontology Research Center (GRC, Baltimore, MD). From weaning (2 Wks), the rats were housed individually in standard plastic cages with beta chip wood bedding. Control animals were fed a NIH-31 standard diet ad libitum (AL). At 1 month of age the calorie restricted (CR) animals were provided a vitamin and mineral fortified version of the same diet at a level of 40% less food (by weight) than AL rats consumed during the previous week. Filtered and acidified water was available ad libitum for both groups. The vivarium was maintained at a temperature of 25°C with relative humidity at 50% on a 12/12-hour light/dark cycle (lights on at 6:00 a.m.) All animals were 6 months of age and sacrificed between 9:00 and 11:00 a.m. The intestine was quickly removed and thoroughly flushed with ice cold PBS and placed into liquid nitrogen then stored at −80°C until processed for Western blotting using standard procedures.

Male Fischer 344 rats (Charles River Laboratories, Wilmington, MA, USA) (approx. 150 g weight) were randomly assigned across groups and, thereafter, administered either vehicle (Veh), or TFBP or TFNBP. Two doses of TFBPs were evaluated: (TFBP: 16.25 mg/kg or 32.5 mg/kg, and for TFNBP: 14.33 mg/kg or 28.66 mg/kg; i.e., equimolar doses), administered by the intraperitoneal (i.p.) route. These compounds were suspended in 1% carboxymethyl cellulose (CMC) in saline (0.9%), and administered 60 min prior to either administration of LPS (1 mg/kg, Sigma, St Louis, MO, E.coli O55:B5 in saline (0.9%), 0.1 ml/kg i.p.) or of Veh (CMC in saline (0.9%) 0.1 ml/kg, i.p.). The selected drug doses are, additionally, equimolar to that of pomalidomide (12.5 mg/kg and 25 mg/kg), which have been demonstrated to be well-tolerated in prior rodent studies, and are of translational relevance to humans [39 (link)]. The i.p. route of drug administration was selected for this first in animal study as it provides 100% bioavailability and, therefore, side steps any caveats associated with potential gastrointestinal bioavailability issues following oral administration.
At 4 h following LPS or Veh, animals were euthanized, and plasma and brain (cerebral cortex) tissue samples were collected and stored at − 80 °C. Brain samples were later sonicated in a Tris-based lysis buffer (Mesoscale Discovery, Gaithersburg, MD, USA) with protease/phosphatase inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, ThermoFisher Scientific, Asheville, NC, USA, diluted to 3X). Next, brain samples were centrifuged (10,000 g, 10 min, 4 °C), and protein concentrations were determined by Bicinchoninic acid assay (BCA, ThermoFisher Scientific, Asheville, NC, USA). An ELISA for TNF-α, IL-6, IL-1β, IL-10 and IL-13 was later performed following the manufacturer’s protocol (Mesoscale Discovery).

Human BM-MSCs were acquired (Cambrex Bioscience Walkersville, Walkersville, MD, USA) and cultured in α-modified minimal essential medium (α-MEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, and 1% penicillin/streptomycin (P/S; Gibco, Carlsbad, CA, USA). We previously characterized naive BM-MSCs. The WB-F344 (rat liver epithelial cells) and WI-38 (human lung fibroblasts) cell lines were purchased from ATCC (American Type Culture Collection, Rockville, MD, USA) and cultured in α-MEM (HyClone) supplemented with 10% FBS and 1% P/S. Each cell line was maintained at 37°C in a humidified atmosphere containing 5% CO₂.