Srt1720

De-identified peripheral blood samples from adults and cord blood were purchased from Research Blood Components, LLC (Boston, MA 02135) and New York Blood Center, Inc. (New York, NY 10065). Erythroid progenitors were cultured in a two-phase system, as previously described.^{29 –31 (link)} Briefly, mononuclear cells from adult peripheral blood (PBMC) or cold blood were isolated on Ficoll-Paque (GE Healthcare, Piscataway, NJ), and cultured in a two-phase culture system.²⁹ The first phase media utilized HyClone™ IMDM (Thermo Scientific, Waltham, MA) containing 30% charcoal treated fetal bovine serum (Life Technology, New York, NY) and rHuIL-3 (25 ng/mL), rHuSCF (50 ng/mL), and rHuEPO at (0.1 u/mL) (GenScript, Piscataway, NJ) for the first 7 days, followed by replacement second phase differentiation media, with 30% charcoal treated fetal bovine serum, rHuEPO at 3 U/mL, and rHuIL-3 (0.1 ng/mL). Test compounds SRT2104 (APExBIO, Houston, TX) and SRT1720 (EMD Millipore, Billerica, MA) were added 4 days after initiation of the Phase 2 erythroid differentiation cultures. Erythroid progenitors were confirmed by Giemsa stain.²⁹ Cells were enumerated by hemocytometry and harvested on day 12 of phase 2 for mRNA and ChIP analysis, and on day 14 for F-cell analysis by flow cytometry and for γ-globin analysis by immunoblotting.

Male C57BL/6J mice (8–12 weeks old) were purchased from Jackson Laboratories (Bar Harbor, ME) and kept in an environmentally controlled room with a 12 h light/dark cycle and ad libitum access to food (LabDiet® PicoLab® Rodent Diet 20, #5053 Purina, Missouri, USA) and water. Mice were i.p. treated with 200 or 300 mg/kg APAP (Sigma-Aldrich) dissolved in warm saline after overnight fasting, and euthanized at the indicated time points between 0 and 96h after APAP injection for collection of blood and liver samples. SRT1720 (EMD Millipore) was dissolved in 10% DMSO plus 2% Tween 20 and was i.p. administered at either 1.5h or 12h and 36h post-APAP. All vehicle control mice received the same volume of DMSO (1 mL/kg) and Tween 20 (0.2 mL/kg). Blood was drawn from the caudal vena cava using a heparinized syringe. The liver was divided into several pieces some of which were used for mitochondrial isolation (Du et al., 2015 ), others for embedding in OCT medium for immunofluorescent staining or fixing in 10% phosphate-buffered formalin for histological analysis. The remaining pieces were snap-frozen in liquid nitrogen and stored at −80°C for later analyses. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University Kansas Medical Center and followed the criteria of the National Research Council for the care and use of laboratory animals.