Fig. 2.

Hypoxia-triggered and HIF1-mediated secretion of HSP90α is essential for hypoxia-induce HK migration. (A) The HIF1α-engineered HKs were cultured in serum-free medium under either hypoxia (1% O₂) or normoxia (20% O₂) for 15 hours. Cell-free conditioned medium (CM) was collected from each culture condition, concentrated and analyzed by western blot analysis with an anti-HSP90α antibody. Equal loading of these samples was based on two parameters: (1) equal volumes of medium were added to each cell culture plate and (2) the volumes of loaded samples were further calibrated according to the cell counts. Recombinant HSP90α (100 ng) was included as the positive control (lane 7). (B) HKs were subjected to colloidal gold migration assays either under normoxia (20% O₂) or hypoxia (1% O₂) in the absence or presence of the indicated concentrations of dimethyl amiloride (DMA) or brefeldin A (BFA) for 15 hours. Only MIs are shown, as previously described. Values are means ± s.e.m. of the MIs from four independent experiments. ^*Statistically significantly different (P<0.03) from the controls under the same experimental condition (i.e. bar 1). (C) HKs were subjected to colloidal gold migration assays under hypoxia or normoxia in the absence (bars 1 and 2) or presence of 5 μg of control IgG (bar 3) or increasing amounts of anti-HSP90α neutralizing antibodies (SPS-771; bars 4-7). Values are means ± s.e.m. of the MIs from four independent experiments. ^*Statistically significantly different (P<0.0%) from their controls under the same experimental condition (bar 1).