Journal: Cell Research

Article Title: A vital sugar code for ricin toxicity

doi: 10.1038/cr.2017.116

Figure Lengend Snippet: 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.

Article Snippet: Ratios of GFP- to mCherry/Cre-expressing cells in the presence and absence of ricin were determined using high-throughput flow cytometry (BD LSRFortessa HTS cell analyzer).

Techniques: Clone Assay, Isolation, Mutagenesis, Inverse PCR, Viability Assay, Expressing, Immunofluorescence, Microscopy, Flow Cytometry

Journal: Cell Research

Article Title: A vital sugar code for ricin toxicity

doi: 10.1038/cr.2017.116

Figure Lengend Snippet: Loss or inhibition of fucosylation confers ricin resistance. (A) Control ( Slc35c1 control ) as well as mutant ( Slc35c1 mut ) 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 Slc35c1 mut and Slc35c1 control 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.

Article Snippet: Ratios of GFP- to mCherry/Cre-expressing cells in the presence and absence of ricin were determined using high-throughput flow cytometry (BD LSRFortessa HTS cell analyzer).

Techniques: Inhibition, Control, Mutagenesis, Light Microscopy, Cell Culture, Alamar Blue Assay, Staining, Flow Cytometry, Expressing

Journal: Cell Research

Article Title: A vital sugar code for ricin toxicity

doi: 10.1038/cr.2017.116

Figure Lengend Snippet: A vital sugar code for ricin toxicity. (A) Proposed sugar code for ricin toxicity. α1,3-fucose residues of Lewis X structures (SSEA-1 (+) ) impair α2,3-sialylation of terminal galactoses (i.e., sialyl Lewis X formation, SSEA-1 (−) ), leading to enhanced exposure of terminal galactoses and ricin binding. Absence of fucosylation allows more efficient sialylation of terminal galactoses and thus is assumed to inhibit ricin binding. GlcNAc (N-acetylglucosamine), Gal (Galactose), NeuNAc (N-Acetylneuraminic acid, one type of sialic acid). (B) Slc35c1 mutant and WT MEFs were stained for α2,3-sialic acid using MALII and counterstained with DAPI to image nuclei. Scale bar, 50 μm. (C) HL-60 cells were treated with the fucosylation inhibitor 2F-peracetyl fucose (FI-1, 100 μM) or the sialylation inhibitor 3F ax -peracetyl Neu5Ac (SI, 250 μM) for 3 days. The amount of the fucose containing, un-sialylated epitope SSEA-1 (CD15) was determined via flow cytometry. SSEA-1 expression of vehicle-treated cells (control) as well as an isotype-matched control (isotype control) is shown. (D) HL-60 cells were pretreated with inhibitors of fucosylation (FI-1, 100 μM) or sialylation (SI, 250 μM) and exposed to different amounts of ricin thereafter. The survival of the cells was determined using Alamar Blue. Data are representative of three independent experiments. (E , F) Slc35c1 wild type (WT) and mutant (KO) mESCs (E) and MEFs (F) were treated with SI (250 μM) and their sensitivity to ricin was assessed using Alamar Blue. Data in D - F are shown as mean ± SD of triplicate cultures. Experiments were repeated three times with similar results. * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant (Student's t -test).

Article Snippet: Ratios of GFP- to mCherry/Cre-expressing cells in the presence and absence of ricin were determined using high-throughput flow cytometry (BD LSRFortessa HTS cell analyzer).

Techniques: Binding Assay, Mutagenesis, Staining, Flow Cytometry, Expressing, Control

Journal: Cell Research

Article Title: A vital sugar code for ricin toxicity

doi: 10.1038/cr.2017.116

Figure Lengend Snippet: 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.

Article Snippet: Ratios of GFP- to mCherry/Cre-expressing cells in the presence and absence of ricin were determined using high-throughput flow cytometry (BD LSRFortessa HTS cell analyzer).

Techniques: Control, Mutagenesis, Flow Cytometry, Clone Assay, CRISPR, Sequencing, Generated, Staining, Infection, Expressing, Construct, Plasmid Preparation

Journal: Nature

Article Title: Comparative glycoproteomics of stem cells identifies new players in ricin toxicity

doi: 10.1038/nature24015

Figure Lengend Snippet: (a) Mixed populations of mutant (KO, GFP+) and their repaired respective wild-type (WT, mCherryCre+) cells were subjected to ricin [4ng/ml] for three days or left untreated (-) and analyzed for viability. The ratios of green to red cells (KO/WT) are shown for each candidate gene. Sister clones for the known ricin sensitivity gene B4Galt131 are shown as positive control and mixtures of GFP and mCherryCre control cells as negative control. (b) HEK cells, harboring CRISPR/Cas9-induced mutations in the indicated genes (2 different sgRNAs each) and control cells (scrambed sgRNAs) were subjected to ricin or left untreated. Cell survival was monitored using Alamar Blue cell viability assay. (c) Igf2r KO murine SCC-VII cells and reconstituted IGF2R wildtype and IGF2R* M6P-binding-domain mutant SCC-VII were subjected to ricin. Cell survival was monitored as above. (d, e) Mixed populations of Igf2r KO and IGF2R/IGF2R* expressing SCC-VII cells were subjected to ricin and the relative amount of IGF2R expressing cells was determined using flow cytometry (d) and confocal microscopy (e). Scale bar 50 μm. Each image is representative of six images of three independent experiments. Data in a, b, c, d are shown as mean ± SD of triplicate cultures. Experiments were repeated three times with similar results. *P<0.05, **P<0.01, ***P<0.001 (two-tailed Student’s t-test).

Article Snippet: Ratios of GFP to mCherry/Cre expressing cells cultured in the presence or absence of ricin were determined using high-throughput flow cytometry (BD LSRFortessaTM HTS cell analyzer).

Techniques: Mutagenesis, Clone Assay, Positive Control, Control, Negative Control, CRISPR, Viability Assay, Binding Assay, Expressing, Flow Cytometry, Confocal Microscopy, Two Tailed Test

Journal: Cancer discovery

Article Title: Human colon cancer-derived Clostridioides difficile strains drive colonic tumorigenesis in mice

doi: 10.1158/2159-8290.CD-21-1273

Figure Lengend Snippet: (A,B) Variation in colonic tumorigenic potential in GF ApcMin/+ mice at 12-14 wk post-inoculation (p.i.) by (A) individual biofilm-positive tumor (BF+T) or (B) biofilm-negative tumor (BF−T) CRC mucosal samples from 6 patients. N = 6-8 mice per group, from 2 independent experiments. (C) 16S rRNA amplicon sequencing relative abundances of the top 20 species in the 6 patient tumor slurry inocula and 12-14 wk p.i. stools from mice gavaged with the tumor slurries. (D) A consortium of 30 bacterial isolates derived from GF mice gavaged with the 3728T slurry recapitulated colonic tumorigenesis of the original 3728T slurry. N = 5-11 mice per group. (E-F) Colonic tumor counts (E) and representative methylene blue staining (F) of colonic tumors (arrows) at 12 wk p.i. in a vancomycin/gentamicin model (see Methods) of chronic colonization with non-toxigenic (TcdA−TcdB−) or toxigenic (TcdA+TcdB+) C. difficile strains. N = 5-18 mice per group, from two independent experiments. (G-H) Colonic tumor counts (G) and representative methylene blue staining (H) of colonic tumors (arrows) from GF ApcMin/+ mice gavaged with the 3728T isolates with or without the CIm_3728T or CIm_3752T C. difficile strains. N = 6-8 mice per group. (I) Colonic tumors induced in GF ApcMin/+ mice gavaged with either the 3979T slurry or the 3979T slurry with C. difficile strain CIm_3728T at 10 wk p.i. Cross represents a mouse death. N = 7-8 mice per group. Statistical significance was calculated via Kruskal-Wallis followed by Mann-Whitney tests. Mann-Whitney p-values are shown.

Article Snippet: Tumorigenesis, high-dimensional flow cytometry, and IHC statistics were performed in GraphPad Prism version 9.1.

Techniques: Amplification, Sequencing, Derivative Assay, Staining, MANN-WHITNEY