Figure 6

Hsp70 is crucial for the pH-dependent transport of DTA across cell membranes into the host cell cytosol. (a) Effect of VER155008 and HA-9 on intoxication of HeLa cells with DT. Cells were pre-incubated for 30 min at 37 °C with HA-9 (10, 20, 50 µM) or VER155008 (VER, 10, 50 µM) or left untreated. Then, DT (6.9 nM) was added and cells were further incubated with DT alone (DT) or with DT and each inhibitor. For control, cells were left untreated (con). After the indicated times, pictures were taken to determine the percentage of rounded cells. Values are mean ± SD (n = 3); significance was tested between cells treated with DT in the absence and presence of inhibitor using Student’s t-test (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001). (b) Less DTA reaches the cytosol of DT-treated HeLa cells pre-treated with VER155008. HeLa cells were pre-incubated for 30 min at 37 °C with either VER155008 (VER, 20 µM), or for control Rad (20 µM). Then, the cells were treated in serum-free medium with nicked biotin-DT (nicked B-DT). For control, cells were incubated with nicked B-DT without inhibitor (nicked B-DT) or left untreated (con). Biotin-DTA from digitonin-extracted cytosolic fractions was detected by Western blotting with streptavidin-peroxidase (Strep) (upper panel). The Western blot panel was cropped for presentation purposes only. The comparable input of cytosolic proteins into the analysis was confirmed by Ponceau S staining (not shown). (c) Effect of VER155008 and HA-9 on the pH-dependent translocation of DTA from cell-bound nicked DT across the cytoplasmic membrane. All cells were pre-incubated for 30 min at 37 °C with 100 nM Baf A1 to block normal uptake of DTA into the cytosol. In addition, cells were treated for 30 min with HA-9 (20 µM), VER155008 (VER, 20 µM) or Rad (20 µM) and incubated in serum-free medium for 15 min at 4 °C with nicked DT (13.8 nM) to allow binding. For control, cells were treated with Baf A1 (con) or with Baf A1 plus nicked DT (nicked DT). Subsequently, all cells were exposed for 10 min at 37 °C to pH 4.5 and further incubated (37 °C, neutral medium, Baf A1). After the indicated times, pictures were taken to determine the percentage of rounded cells. Values are mean ± SD (n = 3); significance was tested between cells treated with nicked DT in the absence and presence of inhibitor using Student’s t-test (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001).