Figure 5. Inhibitors of HSP90 do not affect the viability of monocytes and hMDMs.

(A and B) Monocytes were isolated from PBMCs by elutriation and cultured in media supplemented with 10% autologous serum. (C) hMDMs were differentiated from adherent monocytes for at least 7 d in medium supplemented with 10% HS. Before the experiment, cells were placed in medium supplemented with 1%FCS. (A) Flow cytometry analysis of monocytes labeled with FITC-conjugated annexin V and PI after 5 h culture, with or without the inhibitors of HSP90 Rad, Ge, and GeB (20 µM)or DMAG (1 µM). Dot-plots are from one representative experiment out of 3. The numbers correspond to percentage of cells in quadrants set on the base of autofluorescence. (B) Percentage of TUNEL positive, AnV⁺PI⁻ and AnV⁺PI⁺ monocytes from 3 independent experiments were shown (mean ± sem from 3 independent experiments). Cells treated with S. aureus α-toxin (1 µg/ml) served as positive control. (C) Membrane integrity in Mϕs treated for 5 h with the inhibitors of HSP90 Rad, Ge, and GeB (20 µM) or DMAG (1 µM) was controlled by LDH released into the culture medium (mean ± sem of results from 3 independent experiments). Enzyme released from necrotic (sonicated) hMDMs acted as a positive control.