Hum. Expression of key liver bile acid synthesis genes CYP7A1 and CYP8B1 were 2.5-fold higher with atorvastatin in both strains, but mRNA for liver bile acid export and reuptake transporters and conjugating enzymes were not unaffected. The data suggest that hepatocyte cholesterol and bile acid homeostasis is maintained with combined PCSK9 and HMG-CoA reductase inhibition through efficient liver enzymatic conversion of LDL-derived cholesterol into bile acids and excretion of both, with undisturbed enterohepatic recycling. for 5 min, and for cholesterol 0.5 ml of the upper phase was transferred to a 96-well assay block; for bile acids 0.6 ml of the lower clear layer was transferred to the 96-well block. For cholesterol LC-MS analysis, the dried samples were reconstituted in 200 ul of methanol/well in the 96-well plate, vortexed for 2 min, and centrifuged for 10 min. Supernatant (120 ul) was transferred to new 96-well plates for LC-MS analysis. The LC-MS analysis was performed on a Thermo Acella uHPLC system interfaced with a Thermo Exactive mass spectrometer. The uHPLC column used was a Waters BEH C8 2.1, 1.7 u, 50 mm and the detection was performed in APCI positive ion mode at 25 K resolution and data collection between 200 and 600 Da. For bile acid LC-MS analysis, the dried samples were reconstituted in 200 ul of methanol in the 96-well plate. The plate was vortexed for 2 min and ID1 centrifuged for 10 min. Supernatant (120 ul) was transferred to a new 96-well plate for LC-MS analysis. The LC-MS analysis was performed on a Thermo Acella uHPLC system interfaced with a Thermo Exactive mass spectrometer. The uHPLC column used was a Waters BEH C18 2.1, 1.7 u, 150 mm and the detection was performed in ESI negative ion mode at 25 K resolution and data collection between 200 and 1,000 Da. Liver mRNA extraction and RT-PCR assay At 12 weeks in the mouse study, whole liver was processed for RNA isolation as follows. Tissue samples were immediately placed in RNAlater reagent and kept at 4C for 24 h, then removed and placed at ?80C in cassettes. To prepare RNA, 30 mg tissue samples were added to Biopur tubes with stainless steel bead on dry ice, followed by addition of 1 1.1 ml TRIzol and lysing by TissueLyser at 30 Hz for 3 min. Samples then received 0.4 ml chloroform, were mixed and incubated for 5 min at 20C, and centrifuged for 25 min at 12,000 at 4C. Supernatants (0.35 ml) from each sample were extracted in QIAcubes, and the extracted mRNA samples were stored at ?80C in 96-well plates with 1 l of Protector RNase Inhibitor added. For real-time qPCR measurements, total RNA was quantitated on a NanoDrop ND-1000 UV-Vis spectrophotometer, and RNA quality was assessed on an Agilent BA-53038B 2100 BioAnalyzer. Aliquots of 1 1.0 ug of RNA from each sample were converted to cDNA using the Applied Biosystems BA-53038B (ABI) High-Capacity cDNA Archive Kit. Quantitative real-time PCR was conducted in 384-well reaction plates on an Applied Biosystems Prism 7900HT sequence detector. The mRNA levels for specific genes were normalized to 18S rRNA expression (ABI primer-probe set Hs99999901_s1). Standard BA-53038B curves for each mRNA as well as 18S rRNA were generated by serially diluting cDNA from saline treated animals. All measurements were performed in duplicate and mRNA was averaged within treatment groups (n = 10 mice per group), and unpaired two-tailed 0.05 was considered significant. RESULTS Phenotypic characterization The Y119X point mutation in the PCSK9 coding sequence BA-53038B lies in the prodomain of BA-53038B the protein, analogous to the human.