*2.7. Gene Expression in HSC*

Gene expression was evaluated in primary mHSC on Day 4 or after passage to P1. To assess EV bioactivity, D4 or P1 mHSC were switched overnight to medium containing 2% exosome-depleted serum and then treated for 48 h with 8 μg/mL EV from D4 mHSC, P4 mHSC, or LX-2 hHSC. Total RNA was extracted from the cells using a miRNeasy mini kit (Qiagen) and reverse transcribed using a miScript II RT kit (Qiagen) according to the manufacturer's protocols. Transcripts for common fibrosis-related molecules (e.g., collagen 1a1, cellular communication network factor 2 (CCN2), αSMA) or that corresponded to proteins which were determined by MS to be differentially expressed in EVs from D4 versus P1 mHSC (see below) were evaluated by qRT-PCR using an Eppendorf Mastercycler System and SYBR Green Master Mix (Eppendorf, Hauppauge, NY). Each reaction was run in duplicate, and samples were normalized to 18S ribosomal RNA. Negative controls were a non-reverse transcriptase reaction and a non-sample reaction. Primers are shown in Supplemental Table S1.
