Figure 3
Loss or inhibition of fucosylation confers ricin resistance. (A) Control (Slc35c1control) as well as mutant (Slc35c1mut) HDFs were treated with ricin (4 ng/ml) and L-fucose (10 mM) for 2 days. Their morphology and structural integrity were monitored by light microscopy. Scale bar, 50 μm. (B) Cell viability of Slc35c1mut and Slc35c1control HDFs, supplemented with L-fucose (10 mM) for 24 h and treated with different doses of ricin for 48 h, was determined using Alamar Blue. Data are shown as mean ± SD of triplicate cultures. (C) Toxicity of ricin (after 48-h treatment) in Slc35c1+/+ and Slc35−/− MEFs cultured in the presence or absence of fucose (10 mM), was determined by Alamar Blue assay. Data are shown as mean ± SD of triplicate cultures. (D) Human HL-60 cells were treated with the indicated concentrations of the fucosylation inhibitor 2F-peracetyl fucose (FI-1) for 3 days, stained for SSEA-1 (CD15) and analyzed via flow cytometry. Normal SSEA-1 expression in HL-60 cells (control) as well as isotype-matched control cells (unstained) are shown. (E) HL-60 cells were pretreated with different concentrations of FI-1 and then exposed to different doses of ricin. Survival rates were assessed by Alamar Blue. Representative data of three independent experiments are shown. (F) The number of intact, surviving intestinal organoids was determined and the overall survival with and without the fucosylation inhibitor 2-deoxy-2-fluorofucose (FI-2, 250 μM) was assessed. Data are shown as mean ± SD of triplicate cultures and are representative for three different experiments showing similar results. (G) WT mouse intestinal organoids were pretreated with FI-2 and exposed to ricin (8 ng/ml) for 5 days. Representative images of organoids are shown. Scale bar, 50 μm.
