Asgpr1

iPSC-derived HLCs were treated for 24 h with 5 µM rosuvastatin before the flow cytometry-based LDL uptake assay was performed. iPSC-derived HLCs were dissociated using TrypLE Select (Invitrogen), washed with 1% BSA in PBS, and labelled with primary antibody (ASGPR1; 1:50; Santa Cruz Biotechnology) for 1 h at room temperature. Next, the cells were washed twice with PBS for 5 min each and then incubated for 1 h at room temperature with secondary antibodies (Alexa Fluor 647; 1:500; Invitrogen) diluted in 1% BSA in PBS. Experiments were performed using a Guava EasyCyte Mini Flow Cytometer (Millipore, Billerica, MA, USA). Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).

The following reagents were used: collagenase type IV (Invitrogen, Carlsbad, CA), trypsin inhibitor (Amresco, Cochran Solon, OH), DNase I (AppliChem, Gatersleben, Saxony-Anhalt, Germany), bovine serum albumin (BSA, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), DMEM (Dulbecco's modified Eagle's medium, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), fetal bovine serum (FBS, Hyclone, South Logan, UT), Optiprep^TM density gradient liquid (Axis-shield, Rodelokka, N-0504 Oslo, Norway), ASGPR1 (Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse lgG-PE (Santa Cruz Biotechnology), F4/80 (eBioscience, Santa Clara, California), CD146 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany), APC Rat IgG2b k isotype (BD Pharmingen, San Jose, CA), fluorescein isothiocyanate (FITC) Rat IgG2b k isotype (BD Pharmingen), phycoerythrin (PE) Rat IgG2b k isotype (eBioscience), IC fixation buffer (eBioscience), and permeabilization buffer (eBioscience).