cmi12228-sup-0001-si.pdf158KFig. S1. Rad, CsA and FK506 do not protect HeLa cells from intoxication by B. anthracis PA-delivered His-toxin B1-543. HeLa cells were pre-incubated with Rad, CsA or FK506 (20 μM) for 30 min at 37°C. Subsequently, His-toxin B1-543 (4 μg ml−1) plus PA (2 μg ml−1) were added and cells were further incubated at 37°C. The toxin-induced cell-rounding was determined after 4.5 h. Note, the individual addition of PA or His-toxin B1-543 has no effect on cell morphology (not shown).
cmi12228-sup-0001-si.pdf158KFig. S2. Hsp90-inhibtors Radicicol and 17-DMAG inhibit PTC3 intoxication in a dose-dependent manner. CaCo-2 cells were pretreated with Radicicol (A) or 17-DMAG (B) at the indicated concentration for 1 h. Afterwards, PTC3 (800 ng ml−1 of TcdA1 and 400 ng ml−1 of TcdB2-TccC3) was added and TER values were determined after 5 h. Relative intoxication was calculated in comparison of untreated to toxin-treated cells from three independent experiments.
cmi12228-sup-0001-si.pdf158KFig. S3. Protein loading control corresponding to the Western blot shown in Fig. 3B. Cells were treated exactly as described in the legend to Fig. 3B. Proteins from the cell lysates were separated by SDS-PAGE, blotted onto nitrocellulose membrane and stained with Ponceau S.
cmi12228-sup-0001-si.pdf158KFig. S4. Rad, CsA and FK506 do not inhibit binding of TcdB2-TccC3 to cell-surface bound TcdA1. HeLa cell suspensions (1 × 105 cells in 1 ml PBS/BSA) were incubated without (A) or with 2.0 μg ml−1 of non-labelled TcdA1 for 20 min at 4°C, washed once with PBS/BSA and then incubated without (− Inh) or with the indicated inhibitors (20 μM) together with DyLight488-labelled TcdB2-TccC3 (2.2 μg ml−1, BCDL488) for 10 min at 4°C. Cells were then washed twice in PBS/BSA and applied to FACS analysis. Results are presented as histogram plots, where single cell events are plotted against cell surface-bound fluorescence (Log FL intensity). Grey peaks indicate control cells without protein addition (mock) to obtain the background fluorescence of the cells for comparison. Asterisk indicates unspecific, TcdA1-independent binding of BCDL488 to a small fraction of cells.

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