Figure 6
St3Gal4 determines susceptibility or resistance to ricin. (A) Mixed populations of cells that harbor a reversible gene trap in St3Gal3, St3Gal4 or St3Gal6, as well as parental WT control cells, were subjected to ricin treatment for 3 days. The ratio of mutant (sense, GFP) to WT (antisense, mCherry/Cre) cells was assessed via flow cytometry. Data are shown as mean ± SD of triplicate cultures. (B) Human near haploid KBM7 cells were used to generate ST3GAL4 KO clones using CRISPR/Cas9. Genomic PCR and sequencing of mutagenized clones, as well as control cells, showed appropriately targeted loci (8-bp or 11-bp deletions). Exons are indicated as black boxes. The guide RNAs were designed to delete sequences in exon 6 (arrow). Two different mutant clones were generated (ST3GAL4 KO-1, KO-2). (C) ST3GAL4 mutant and control human KBM7 cells were stained for sialyl Lewis X (CD15s) and analyzed via flow cytometry. (D) ST3GAL4 KO-1 and KO-2 KBM7 cells, as well as control cells, were treated with different amounts of ricin and their viabilities were determined. Data are representative of three independent experiments. (E, F) mESCs were infected with a doxycycline-inducible expression construct coding for ST3GAL4 together with mCherry, or an empty control vector coding for mCherry only. (E) Mixed populations of infected and uninfected, as well as control cells, were treated with doxycycline and exposed to ricin (4 ng/ml) for 9 days. The percentage of mCherry+ cells was monitored over time by flow cytometry. Data are shown as mean ± SD of triplicate cultures. (F) An ST3GAL4-overexpressing mESC clone as well as an empty vector control were treated with various concentrations of ricin and their viabilities were determined using Alamar Blue staining. Data are shown as mean ± SD of triplicate cultures.
