A–D. Cultured cholangiocytes were pre-treated with either methyl-β-cyclodextrin (Mβ-cd) or ASM-siRNA and then exposed to C. parvum sporozoites followed by dual labelling fluorescence with a polyclonal antibody to C. parvum and FITC-tagged PNA lectin to label Gal/GalNAc epitopes as we previously reported (Chen and LaRusso, 2000). The left lower insets in (A)–(C) show labelling of Gal/GalNAc in the boxed regions. (A) Untreated cells showed strong labelling of FITC-tagged PNA lectin at infection sites [arrowheads in (A) and inset]. Cells treated with 10 mM Mβ-cd for 1 h prior to C. parvum infection did not show strong labelling of FITC-tagged PNA lectin at infection sites [arrowheads in (B) and inset]. Cells transfected with Cy3-tagged ASM-siRNA showed a significant decrease in Gal/GalNAc accumulation at infection sites [arrowheads in (C) and inset], whereas non-transfected cells showed strong Gal/GalNAc accumulation [arrow in (C)]. Cells transfected with ASM-siRNA were identified by positive fluorescence of Cy3 [as outlined in (D)]. Bar = 5 µm.
E. Quantitative analysis as determined by number of sites with positive accumulation per 100 infection sites. *P < 0.05, compared with control. Scr-siRNA, scrambled siRNA.