Results were normalized to total protein concentration. RNA was extracted using the Qiagen RNeasy Mini Kit (Valencia, CA) or Trizol reagent (Sigma-Aldrich), and cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad). Gene expression was quantified using iTaq Fast SYBR Green Supermix with ROX (Bio-Rad) and gene-specific primers (Invitrogen, Coralville, IA) listed in Supporting Table 1, or TaqMan Mm00627280_m1 (tnfaip3), Mm00607939_s1 (β-actin). Expression of target
genes was normalized to that of the housekeeping genes β-actin, TATA box binding protein (TBP), or 28S. MicroRNA (miRNA) was extracted using the mirVana kit (Life Technologies, Grand Island, NY), and assayed for miR203, Ensartinib and the housekeeping miRNA, snoRNA202, using TaqMan (Applied Biosystems, Foster City, CA). qPCR
were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems). We generated recombinant adenovirus (rAd).A20 using a plasmid provided by Dr. V. Dixit (Genentech, San Francisco, CA).24 The rAd.βgal was a gift of Dr. Robert Gerard (University of Texas SW, Dallas, TX). By RT-PCR, we generated HA-tagged deletion mutants comprising the N-terminus (Nter) and seven Zinc (7Zn) domains of A20 and cloned them in pAC CMVpLpA SR(+) expression plasmid to generate rAd. (Supporting Methods). We used HEK293 cells to generate, produce, and titer CT99021 solubility dmso rAd. that were purified by cesium chloride density gradient centrifugation for in vivo,24 or the AdenoPure LS Kit (Puresyn, Malvern, PA) for in vitro experiments. Hepatocyte cultures (60% confluent) were transduced with rAd. at a multiplicity of infection (MOI) of 50-200 plaque-forming units per cell (pfu/cell), leading to transgene expression in >95% of cells without toxicity14, 15 (Supporting Fig. S1). selleck kinase inhibitor In vivo, we injected 1 × 109 pfu of rAd. in 100 μL saline into the mouse penile vein. This dose and route of administration achieves maximal transgene expression in 30% of hepatocytes, 5 days after injection.15 Transgene expression was analyzed by WB (A20) and X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) staining (β-gal). A 78%
hepatectomy (EH) was performed as described.15 Livers harvested before and after surgery were either frozen in liquid nitrogen for protein and RNA extraction, or fixed in 10% formalin for immunohistochemistry (IHC) and immunofluorescence (IF) analysis. For IHC and IF staining we used the following primary antibodies: goat anti-SOCS3, rabbit anti-P-STAT3 (Cell Signaling), rat anti-Ki67 (Dako), chicken anti-albumin (Novus Biologicals, Littleton, CO), and goat anti-HNF4α (Santa Cruz), followed by horseradish peroxidase (HRP) or Alexa Fluor 488 (green) and 594 (red) conjugated secondary antibodies (Invitrogen, Carlsbad, CA). Ki67, P-STAT3, and SOCS3-positive cells per high-power field (HPF) were counted using ImageJ automated or manual cell counting.