Figure 6. Hsp90 inhibition attenuates TGF-β–induced myofibroblast transformation and ECM gene expression.

(A) Primary lung-resident fibroblasts (CD45^–Col1⁺) were isolated from lung cell cultures of αSMA^creERROSA^mTmG mice and treated with TGF-β and 4-hydroxy tamoxifen in the presence and absence of 17-AAG for 72 hours. Immunofluorescence images were obtained at an original magnification of ×10. Scale bars: 200 μm. (B) The number of GFP-positive and total DAPI-positive (blue) cells were quantified using MetaMorph image analysis software (n = 4–5/group) and are indicated as the percentage αSMA-positive cells in total DAPI-positive cells. Data are cumulative of 2 independent experiments with similar results. (C and D) Human lung-resident fibroblasts (CD45^–Col1⁺) were isolated from non–idiopathic pulmonary fibrosis (non-IPF) lung fibroblast cultures and treated with TGF-β in the presence and absence of 17-AAG for 24 hours. Transcripts of extracellular matrix (ECM) genes COL1α and FN1 were quantified using qRT-PCR and are shown as the fold-induced gene transcripts relative to GAPDH. Data are representative of 2 independent experiments with similar results (n = 4–5/group). (E) Lung-resident fibroblasts of αSMA^creERROSA^mTmG mice were transfected with control, Hsp90AA-specific, or Hsp90AB-specific siRNA. After a 24-hour transfection, cells were treated with TGF-β and 4-hydroxy tamoxifen for 72 hours and the number of GFP-positive and total DAPI-positive cells were quantified using MetaMorph image analysis software (n = 4–5/group). Data are cumulative of 2 independent experiments with similar results. One-way ANOVA with Sidak’s multiple comparisons test was used to measure significant difference for all the experiments. All data shown are mean ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005. ns, not significant.