Figure 1
Loss of Slc35c1 and Fut9 protects cells from ricin toxicity. (A) Randomly mutagenized single-cell mESC clones were exposed to ricin (2 ng/ml) for 10 days and ratios of GFP+/mCherry+ cells were measured. Isolated, ricin-resistant, mutant clones were then analyzed via inverse PCR and their integration sites were determined. All clones were found to harbor the gene trap in sense orientation at the indicated intronic sites (green arrows) of either Fut9 (asterisks) or Slc35c1 (black triangles). (B) Survival of mESCs harboring a gene trap in either Fut9 or Slc35c1 in sense (KO) or antisense (WT) orientation in the presence of the indicated concentrations of ricin. Alamar Blue cell viability assay was used to determine cell survival. Data are representative of three independent experiments. (C) Independent Fut9 and Slc35c1 mutant (KO) and reverted WT mESC sister clones were grown in the presence or absence of ricin (8 ng/ml). Representative images are shown. Scale bar, 100 μm. (D) Mixed populations of unlabeled Slc35c1 WT and mutant (KO) mESCs were exposed to different concentrations of ricin for 3 days. The amount of fucose (detected by AAL) and Lewis X (SSEA-1, CD15) expressing cells was monitored by immunofluorescence microscopy (upper panels) and flow cytometry (lower panels). Scale bar, 50 μm.
