Figure 2

Slc35c1 mutant MEFs and intestinal organoids show increased resistance to ricin. (A) Slc35c1 WT and KO MEFs were cultured in the presence or absence of ricin. Cell survival was determined after 3 days by Alamar Blue. Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (Student's t-test). (B) Representative images of Slc35c1^+/+ and Slc35c1^−/− MEFs in the presence or absence of ricin (10 ng/ml) for 2 days. Scale bar, 50 μm. (C) WT and Slc35c1 mutant MEFs were stained for the presence of fucose-containing glycans using AAL and microscopically analyzed. Scale bar, 25 μm. (D) Murine intestinal organoids isolated from control Slc35c1^+/+ and mutant Slc35c1^−/− mice were treated with ricin (8 ng/ml) for 5 days and the numbers of surviving, intact organoids after ricin exposure were assessed. The percentage of surviving organoids compared to untreated gut organoids was determined. Data are shown as mean ± SD (n = 3). Representative data from five different experiments are shown. (E) Slc35c1^+/+ and Slc35c1^−/− intestinal organoids were treated with different doses of ricin for 5 days and stained for markers (Epcam to detect epithelial cells; AAL to detect fucosylated glycans; and UEA to specifically detect α1,3-fucosylated glycan structures) to assess organoid integrity. DAPI was used as a nuclear counterstain. Representative images are shown. Scale bar, 50 μm.