A, Senescence-associated β-galactosidase activity (SA-β-gal) assay; bystander normal human cholangiocytes (NHCs) were incubated with normal and senescent (lipopolysaccharide [LPS]-induced, and primary sclerosing cholangitis [PSC]-derived) cholangiocyte-derived EVs (1e8/ml) every other day for 10 days. SA-β-gal activity was assessed using β-galactosidase assay. Arrows indicate representative SA-β-gal positive NHCs. B, Quantification of the SA-β-gal assay; data presented as percentage of SA-β-gal positive cells per total cell count. The PSC patient-derived data represent the average of 3 replicates of 3–5 separate PSC patient samples. C, Induction of senescence was assessed by measuring messenger RNA (mRNA) expression (RT-qPCR) of various cellular senescence markers. Bars represent mean (±) standard error of the mean (SEM); n=3–5. *P<0.05, **P<0.01, #P=0.07.