Figure 3. Mechanisms of Hsp90-driven migration and invasion in fibroblasts.

Primary lung fibroblasts (CD45^–Col1⁺) isolated from lung fibroblast cultures of human idiopathic pulmonary fibrosis (IPF) or TGF-α–transgenic (TGF-α–Tg) mice on doxycycline (Dox) for 4 weeks by negative selection with anti-CD45 magnetic beads. (A) Quantification of migration of IPF fibroblasts treated with vehicle or 17-AAG (1 μM) for 24 hours (n = 6/group). (B) Quantification of invasiveness of IPF fibroblasts treated with vehicle or 17-AAG (1 μM) for 24 hours (n = 6/group). (C) Fibroblasts of TGF-α–Tg mice on Dox for 4 weeks were transiently transfected with control, Hsp90AA-specific, or Hsp90AB-specific siRNA for 48 hours and the migration was quantified for 24 hours (n = 6/group). (D) Costaining of F-actin and Hsp90 in lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks. Hsp90 colocalized (white signal) with F-actin in cytoplasmic fibrillary adhesions or focal complexes. Images were obtained at an original magnification of ×40. Scale bars: 40 μm. (E) F-actin stained using phalloidin in lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks treated with vehicle or 17-AAG (1 μM) for 24 hours. Scale bars: 40 μm. (F) Immunoblot analysis for FAK, p-Tyr^576/577FAK, ERK, p-Tyr⁴⁶⁸ERK, cell division control protein 42 homolog (Cdc42), and Ras homolog gene family, member A (RhoA) in the lysates of lung fibroblast of TGF-α–Tg mice on Dox for 4 weeks treated with vehicle or 17-AAG (0.5 μM) for 48 hours. (G) The total expression and phosphorylation of the indicated proteins were measured and normalized to GAPDH levels in the total lysates (n = 3/group). (H) Quantification of Ccl3, Ccl20, Il-10, and Ccr5 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.5 μM) for 24 hours. (I) Quantification of Has2, Cd44, Mmp12, and Timp1 gene transcripts relative to Hprt in the lung fibroblasts of TGF-α–Tg mice on Dox for 4 weeks and treated with vehicle or 17-AAG (0.5 μM) for 24 hours (n = 3/group). Results are representation of 3 independent experiments with similar results. Data shown are mean ± SEM. An unpaired 2-tailed Student’s t test was performed to measure the significance. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005.