Age-matched 6 to 9-week-old male mice were used for all experiments. Sources for the different strains of mice can be found in the Supporting Material. All experiments were performed IWR-1 ic50 in accordance with guidelines from the University of California, Los Angeles Institutional Animal Care and Use Committee. For ASA-induced

toxicities, mice were given ASA (180 mg/L, Sigma-Aldrich) in drinking water for 5 days. For APAP hepatotoxicity studies, mice were fasted for 18 hours and then administered vehicle (normal saline, 0.9% NaCl) or APAP (175-600 mg/kg, Sigma-Aldrich) by intraperitoneal injections (i.p.). For serum and histological studies, mice were sacrificed at 6-7 hours post-APAP administration and serum and liver samples were retrieved. For polyI:C and VSV studies, mice were injected with saline or polyI:C (100 μg, Invivogen) or VSV (2.5e7 plaque-forming units [pfu]) i.p. 24 hours prior

to APAP treatment. Green fluorescent protein (GFP)-tagged VSV was a kind gift from G. Barber (University of Miami, Miami, FL). To study the effects of pregnenolone 16α-carbonitrile (PCN) on APAP treatment, mice were injected (i.p.) with PCN (75 mg/kg, Sigma-Aldrich) or control (1% DMSO, corn oil) 24 hours prior to APAP treatment. To study the effects of ethanol (EtOH) on APAP treatment, mice Selleck ACP-196 were given 20% EtOH (Gold Shield Chemical) in water ad libitum for

5 days prior to APAP administration. PolyI:C treatment for these experiments occurred at days 3 and 5. Serum alanine aminotransferase (ALT) levels were determined using the manufacturer’s protocol (TECO Diagnostics). For hematoxylin and eosin (H&E) staining, liver samples were fixed in formalin for 48 hours. H&E staining was performed at the UCLA Tissue Procurement Core Laboratory (TPCL). APAP-protein adduct staining was done using anti-APAP rabbit antibodies (Biogenesis/AbDserotec) as described.23 For quantitative real-time PCR (Q-PCR), total liver RNA was isolated and cDNA was synthesized according medchemexpress to the manufacturer’s protocol: Trizol (RNA) and Bio-Rad iScript (cDNA). PCR reactions were set up using Quantise Master Mix (2X SensiMix SYBR and Fluorescein). Mice were given ASA (180 mg/L) in their drinking water for 5 days. Mice that were infected with VSV (2.5e7 pfu) at day 1 experienced higher ASA-induced hepatotoxicity compared to uninfected mice. This is evidenced by higher levels of serum ALT in mice treated with VSV and ASA compared to the ASA only group (Fig. 1A). Histological analysis of livers from VSV-infected mice further evidenced hepatic injury as indicated by increased fat (steatosis) on oil-red staining (data not shown) as well as H&E staining sections (Fig. 1B).