Figure 7.

eHSP90α had a stimulatory effect on HPDE cell anchorage independence but not cell proliferation. (A) Growth curves of HPDE cells treated with control medium (CTRL) or MϕCM for 1 to 5 days in the presence of control IgG or anti-CD91 antibody. Trypan blue exclusion assay was performed to count the numbers of viable cells. The data represent the averages of three independent experiments, and each experiment was performed in triplicate. (B) Growth curves of HPDE cells treated with PBS or 15 μg/ml of rHSP90α for 1 to 5 days. Trypan blue exclusion assay was performed, and the averages of three independent experiments are shown. (C) Flow cytometric analyses of the cell-cycle phase distribution of control medium (CTRL)- vs. MϕCM-treated HPDE cells (upper panel) and PBS- vs. rHSP90α-treated HPDE cells (lower panel). HPDE cells were treated for 24 h and trypsinized for ethanol fixation and propidium iodide staining. The histograph of cell-cycle phases was obtained from 10000 cells analyzed using a FACSCalibur flow cytometer. (D) The levels of cyclin D1, CDK4, Ser-780-phosphorylated Rb, Rb, cyclin E, cyclin A, Tyr-15-phosphorylated CDK2, CDK2, cyclin B, Tyr-15-phosphorylated CDC2, CDC2, and GAPDH in HPDE cells treated with PBS, 15 μg/ml rHSP90α, or 15 μg/ml rHSP90α plus preimmune IgG or anti-CD91 antibody. (E) Soft-agar colony-forming assay of PBS or rHSP90α-treated HPDE cells. HPDE cells (2000 cells seeded on the soft-agar layer in each well of a 6-well plate) were treated with or without 15 μg/ml rHSP90α and refreshed every 3 days for 3 weeks. The colonies were stained with Giemsa and photographed under a microscope (100 ×); only colonies consisting of ≥80 cells were counted. The mean ± SD values of three independent experiments are shown, and each experiment was performed in triplicate. ^#, P < 0.05 when compared with the data of PBS-treated cells. (F) Phosphorylated FAK and total FAK levels in HPDE cells treated with PBS, rHSP90α, or rHSP90α plus control IgG or anti-CD91 antibody.