C - Michigan Technological University

weeks of hibernation, and 3 bears were sacrificed after at least 14 weeks of physicalactivity following hibernation. Bears were euthanized by an injection of pentobarbital (10ml per 100 lbs body weight). Transiliac crest biopsies were collected with a diamondedged3/8” coring bit. The cortical sections were separated from the trabecular bone witha saw. Marrow was removed from the trabecular bone with a water pick. Cortical andtrabecular sections were crushed separately into a fine powder in liquid nitrogen, andlysed in buffer L (10mM Tris-HCl pH 8.0, 1% IGEPAL, 2mM MgCl 2 , 150mM NaCl). Aftercentrifugation to remove insoluble matter, protein concentration was quantified with theDC Protein Assay (BioRad Laboratories, Hercules, CA). Aliquots were stored at -80 o C.15 μg of lysate was brought up to 50 μL in buffer L, and 50 μL of chemiluminescentDEVD substrate was added. After 1 hour incubation, caspase-3/7 activity was quantified.Caspase activity was normalized to a standard curve of purified caspase-3 (Enzo LifeSciences, Plymouth Meeting, PA). Caspase-3/7 activity was reported in internationalunits (U) of activity. One U breaks down DEVD substrate at 1 pmol per minute.5.2.5 MC3T3-E1 gene expression response to bear serumCells were seeded into 6-well plates at 20,000 cells/cm 2 and grown overnight.Osteoblast differentiation was cultured for 6 days in differentiation media (α-MEM with2% bear serum, 50 μg/mL ascorbic acid and 10 mmol β-glycerophosphate), with a mediachange on the third day. RNA was purified with the SV Total RNA Isolation System(Promega, Madison, WI). Immediately after bear serum stimulation, media wasaspirated, and cells were directly lysed in 140 μL RNA lysis buffer. Quality of RNA wasassessed by gel electrophoresis and with a Nanodrop (Nanodrop Technologies,Willmington, DE). cDNA equivalent of 300 ng RNA was generated in a 25 μL reactionusing 250U SSII reverse transcriptase, 0.5 μg oligo-dT 12-18 primer, and 25 U RNaseOut (Life Technologies, Carlsbad, CA) at 42°C for 20 minutes, 50°C for 10 minutes and42°C for 1 hour in a gradient thermocycler (Eppendorf, Westbury, NY). Primers for allgenes of interest and the housekeeping gene were designed using PrimerQuestsoftware (Integrated DNA Technologies, Coralville, IA) and the NCBI gene banksequences, and the PCR conditions for each primer set were optimized using RNA fromMC3T3-E1 cells. SYBR Green 2X master mix (ABGene, Rochester, NY) was used to setup PCR. cDNA equivalent of 2.5ng RNA was used in 25 μL PCR samples with 0.1 μM74