Animals were allowed food and water ad GSK2118436 datasheet libitum. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Hepatocytes were isolated by adaptation of the calcium two-step collagenase perfusion technique as described.15, 17 Hepatocytes were plated on collagen-coated six-well plates
(BD Biosciences, San Jose, CA) at 250,000 cells/well. After the initial 2-hour attachment period, plating media was changed to either HGM complete with growth factors (+GF) HGF (supplemented at 40 ng/mL) and EGF (supplemented at 20 ng/mL) or without growth factors (−GF) and every 48 hours thereafter. Cells were harvested Birinapant clinical trial on day 0 (2-hour plated), 2, 4, 6, 8, and 10 for RNA and protein. Total RNA was extracted from plated cells using the RNABee
reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The isolated RNA was treated with Turbo DNA-free (Ambion, Austin, TX) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry at 260 nm and purity was assessed by optical density 260/280 ratio. The RNA was stored at −80°C. The experiment was repeated in three rats and their messenger RNA (mRNA) was pooled for further processing in primary hepatocytes as well as 70% partial hepatectomy (PHx) experiments. Four micrograms RNA per sample was reverse-transcribed using random hexamer to complementary DNA (cDNA) by using SuperScriptIII reverse transcriptase (Invitrogen, La Jolla, CA) according to the manufacturer’s protocol. A no reverse transcriptase (RT) control was also
included. The gene-specific primers used for rat were as follows. REST, cMyc, Klf4, Nanog (SuperArray Bioscience, Cat. no. PPR45101A, PPR45580A, PPR43919A, and PPR59663A, respectively). Oct4 forward: 5′-GGC GTT CTC TTT GCA AAG GTG TTC-3′; Oct4 reverse: 5′-CTC GAA CCA CAT CCT TCT CT-3′. Expression levels of REST, Oct4, cMyc, and Klf4 were determined by qRT-PCR using SYBR green and levels were normalized relative to expression of MCE cyclophilin in each sample. Fold change in gene expression was calculated by using the 2(−ΔΔCt) method.18 Reverse-transcribed samples were amplified in parallel on an ABI prism 7000 SDS instrument (Applied Biosystems, Foster City, CA). qRT-PCR for each sample was performed in triplicate in a 20-μL reaction with 50 ng of cDNA, 5 picomoles of each primer, and 1× SYBR green PCR mix (Applied Biosystems). The standard conditions for real-time PCR were as follows: 2 minutes at 50°C, 10 minutes at 95°C followed by 40 cycles of 15 seconds denaturation at 95°C, and elongation at 60°C for 45 seconds. A dissociation curve analysis was performed at the end of every run. A no RT and a no template control were also included in every run.