Figure 5.

The assistance of AIP56 intoxication by Hsp90 and cyclophilins involves the catalytic domain of AIP56. (A) Pharmacological inhibition of Hsp90 and cyclophilins do not affect the cellular uptake of Bla mediated by the AIP56 B domain. mBMDM were pre-treated with 17-DMAG + Rad or CsA, incubated Bla-AIP56^258–497 for 15 min at 37 °C and loaded with CCF4-AM for 30 min. Untreated cells and cells treated only with the inhibitors were used as controls. Uncleaved and cleaved CCF4-AM were detected by microscopy and the ratios determined by quantifying a minimum of 10 microscopic fields for condition. Results shown represent one out of three independent experiments. Statistical significance between mean increments in the ratio of cleaved/uncleaved CCF4-AM induced by Bla-AIP56^258–497 in the presence or absence of the drugs was tested by the t-test and Mann-Whitney test (n = 3 independent experiments) and no difference was observed. (B) Cellular uptake of LF_NAIP56^1–261 is prevented by pharmacological inhibition of Hsp90. mBMDM were pre-treated with Rad, 17-DMAG, CsA or FK506, alone or combined, and intoxicated with LF_NAIP56^1–261 + PA in the presence of the inhibitor(s). Cleavage of p65 was detected by western blotting (chromogenic detection). A representative blot is shown (the full blot is presented in Fig. S7). The box-plot shows the quantification of the blots (n = 6 independent experiments). Loading correction was achieved by dividing the density of p65 by the respective density of α-tubulin. Statistical significance was tested by one-way ANOVA. p-values for individual comparisons were calculated using the Dunnett’s test and refer to comparisons to cells treated only with toxin.