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. 2009 May 22;284(32):21684–21695. doi: 10.1074/jbc.M109.006254

FIGURE 1.

FIGURE 1.

Screening of ricin-resistant clones generated by EMS mutagenesis. A, ricin resistance of ricin-selected cell lines. Individual cell lines were treated with graded doses of ricin for 4 h, and their subsequent ability to synthesize proteins was determined. Blue, parental HEK 293T (n = 24); open circles and dotted lines, HEK 293S Lec1 (n = 4); gray (n = 1–4), orange (n = 1, 2, 3, or 6), violet (n = 1–3), and green (n = 6 or 7) indicate cell lines belonging to various selection screen-defined classes. Error bars have been omitted for clarity. B, nonselected sensitivity of all of these cell lines to PEx. Cell lines were treated with graded doses of PEx for 4 h, and their subsequent ability to synthesize proteins was determined. Blue (n = 10), open circles (n = 2), gray (n = 1–4), violet (n = 1 or 3), and green (n = 3) are same as in A; orange (n = 1–5) indicates cells with ∼5-fold or greater resistance to PEx. Error bars have been omitted for clarity. C, scatter plot of PEx resistance versus ricin resistance for all of the cell lines generated. Sensitivity of the cell lines to toxin was determined as described in A by measuring the concentration (IC50, ng/ml) of toxin required to reduce protein synthesis to 50% of that of untreated cells, and resistance (-fold) was calculated from the ratio of the IC50 for the cell line versus the IC50 for HEK 293T cells. PEx-resistant cells (orange) cluster as a group with 2–9-fold ricin resistance; green, a cluster of cell lines with very high ricin resistance. D, scatter plot of relative number of ricin B chain-binding sites versus ricin resistance for 62 of the cell lines generated (7 “gray” and 1 “green” cell line were not tested). The relative number of ricin-binding sites on the cell lines was determined by flow cytometry using Alexa 647-labeled ricin B chain. Apart from the highly ricin-resistant lines (green), the cell lines cluster into two broad groups, with either medium to high (group I) or low to medium (group II) numbers of ricin-binding sites. Violet-colored symbols, candidate HEK 293T Lec36 cells.