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. 2017 Apr 3;7:45843. doi: 10.1038/srep45843

Figure 7. Neutralization of Stx1 cytotoxicity with S-hyIgA plantibodies in vitro.

Figure 7

Stx1 holotoxin (10 or 100 pg/mL) was pre-incubated with a sample from secretory Tg plants, dimer Tg plants, or wild-type plants (50%-saturated ammonium sulphate-precipitated preparation of leaf extract), or with purified mouse IgA myeloma protein TEPC 15. The mixture was added to butyrate-treated Caco-2 cells (Stx1 100 pg/mL) or untreated Vero cells (Stx1 10 pg/mL). After cells had been cultured for 48 h, the cytotoxicity of Stx1 was evaluated by the following assays. (a,b) Viability of Caco-2 cells (a) or Vero cells (b) was determined by WST-8 assaying. The symbols represent secretory Tg plants (open circles), dimer Tg plants (open squares), wild-type plants (open triangles), or IgA myeloma TEPC 15 (open diamonds). The viability (ordinate) is relative to that of toxin untreated cells. Data are expressed as means ± SD of triplicate determinations. Error bars underneath the symbols are not visible. The doses of antibodies are expressed as IgA concentration or TSP (abscissa), as shown in Fig. 5(c). Apoptosis of butyrate-treated Caco-2 cells was detected as DNA fragmentation. After exposure to Stx1 (100 pg/mL), the DNA fragmentation was visualized by agarose gel electrophoresis. Lane 1, untreated; lane 2, Stx1-treated; lane 3, Stx1 + WT-treated (TSP 0.6 mg/mL); lane 4, Stx1 + TEPC 15-treated (1 μg/mL IgA); lane 5, Stx1 + secretory Tg (0.1 μg/mL IgA); lane 6, Stx1 + secretory Tg (1 μg/mL IgA); lane 7, Stx1 + dimer Tg (0.1 μg/mL IgA); and lane 8, Stx1 + dimer Tg (1 μg/mL IgA).