Alzet mini osmotic pumps model 2006

Synthetic human IMD_1-53 was from Phoenix Pharmaceuticals (Burlingame, CA, USA). Alzet Mini-osmotic Pumps (model 2006) were from DURECT Corp (Cupertino, CA, USA). Primary antibodies for IMD (sc-86272), β-actin (sc-47778), IL-1β (sc-7884), and all horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies for CD68 (ab125212), α-actin (ab5694), glucose-regulated protein78 (GRP78, ab21865), activating transcription factor (ATF4, ab216839), cleaved ATF6 (ab203119), p-inositol-requiring kinase 1 alpha (p-IRE1α, ab48187), CHOP (ab10444), cleaved-caspase-3 (ab13847) were from Abcam PLC (Cambridge, UK). Apoptosis-associated speck-like protein containing CARD (ASC, SAB4501315) were from Sigma-Aldrich (St. Louis, MO, USA). Dylight-labeled secondary antibodies were from EarthOx Life Sciences (Millbrae, CA, USA). The kit for reverse transcription of RNA and SuperReal PreMix Plus for real-time PCR (TIANGEN Biotech, Beijing, China). The oil red O and taurine (Tau) were from Sigma-Aldrich (St. Louis, MO, USA). Oxidized human LDL (ox-LDL, YB-002) was from Yiyuan Biotechnology (Guangzhou, China). Other chemicals and reagents were of analytical grade.

PRI-724 (PRISM Pharma Co., Ltd.) was diluted in phosphate-buffered saline (PBS) and injected intraperitoneally once daily at a concentration of 5 or 20 mg/kg body weight for 6 weeks. For the 1 week ON/OFF experiment, the reagent (5 mg/kg/day) was administered intraperitoneally for 6 weeks during which animals were treated for a total of three cycles, each consisting of 1 week of once-daily dosing followed by 1 week without dosing. In a separate experiment, PRI-724 was administered intraperitoneally at 15 mg/kg body weight at a frequency of once or twice per week for 6 weeks. Alternatively, PRI-724 was dosed by subcutaneous infusion (1 mg/kg/day) for 6 weeks using implanted ALZET Mini-osmotic pumps MODEL 2006 (DURECT, Cupertino, CA). On the last day of the respective treatment, each animal was humanely killed and the liver was recovered at necropsy. Separate segments of each animal’s liver were fixed in formalin (for standard histology and immunohistochemistry); embedded in optimal cutting temperature (OCT) compound and frozen at −80 °C (for cryosectioning followed by immunohistochemistry); or flash-frozen in liquid nitrogen (for analysis of hydroxyproline content and assays of RNA and protein expression).