E88 129

Human apoB levels were determined using a commercial sandwich ELISA (3715-1H-6; Mabtech, Cincinnati, OH), and the signal was detected using the HRP-TMB system. The concentration of apoB was calculated using a standard curve with a four-parameter logistic regression model. Human apoA1 levels were determined by ELISA (3710-1H-6; Mabtech) following the manufacturer’s instructions. Human Albumin levels in cell culture supernatants were measured by commercial ELISA (E88-129; Bethyl Laboratories, Montgomery, TX) following the manufacturer’s instructions. Lp(a) levels were measured using the Human Lipoprotein A SimpleStep ELISA kit (ab212165; Abcam, Cambridge, UK) as described by the manufacturer. Mouse serum apoB levels were measured using the Mouse ApoB Simplestep ELISA Kit as described by the manufacturer (ab230932; Abcam, Cambridge, UK). Total cholesterol and LDL/VDL levels were measured in humanized mice using the Cholesterol Assay Kit - HDL and LDL/VLDL (ab65390; Abcam, Cambridge, UK) and triglyceride levels measured using Triglyceride Assay Kit, Quantification (ab65336; Abcam, Cambridge, UK).

Cell culture media was collected every time media was exchanged (Figure 1) and stored frozen at −80 °C until analyses for various biomarkers, including albumin, urea, lactate dehydrogenase (LDH), and human interleukin-6 (IL-6). The ELISA assays for albumin (E88-129, Bethyl Laboratories, Montgomery, TX, USA), urea (EIABUN, ThermoFisher), LDH (ab102526, Abcam, Cambridge, UK), and IL-6 (D6050, R&D Systems) were conducted using manufacturer’s instructions. For CYP3A4 activity, the P450 Glo 3A4 with Luciferin-IPA assay (V9001, Promega, Madison, WI, USA) was used; this is a live cell assay, and it was conducted according to the manufacturer’s instructions as follows. The Luciferin-IPA substrate was diluted 1:1000 in maintenance media and added to chips or wells 1 h prior to reading luminescence using a plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA). After the assay, the media was replaced.