qPCR analysis

PSC or HLC were cultured overnight in 5% LPDS (Thermo Fisher Scientific) medium alone or supplemented with either 5 µM rosuvastatin (EMD Millipore, Burlington, MA, USA) or excess sterols (10 µg/ml cholesterol and 5 µg/ml 25-hydroxycholesterol; Sigma-Aldrich). Positive control cells were treated with 5 µg/ml TM (Thermo Fisher Scientific) for 4 h or FBS (Thermo Fisher Scientific) overnight. Control cells were treated with DMSO overnight. At the end of treatment, PSC and HLC were lysed using 150 µl of 0.1% β-mercaptoethanol in RLT Buffer (Qiagen, Valencia, CA, USA). The lysates were purified with Qiashredder and RNeasy kits (Qiagen) according to the manufacturer's instructions. RNA was quantified with a NanoDrop One Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using 1 µg RNA with SuperScript IV reverse transcriptase (Thermo Fisher Scientific) in a 20 µl volume. qPCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific) with primers obtained from Integrated DNA Technologies (Table S1). Reactions were run on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Raw data were quantified in Microsoft Excel and statistics performed in GraphPad Prism 8 (La Jolla, CA, USA). PCR was carried out for spliced XBP1 and XBPI expression with PCR Supermix (Thermo Fisher Scientific). Amplicons were evaluated via 2% agarose gels (Bio-Rad, Hercules, CA, USA). Then, 10 µl of amplicons were added to 2 µl 6× loading buffer (Thermo Fisher Scientific). Gels were run at 80 V for 60 min and imaged via a ChemiDoc Imaging System with Image Lab Touch Software (Bio-Rad).