Figure 7. Induction of autophagy by inhibition of VimIF assembly decreases invasiveness.
Stable transfection with mEmerald-vimentin construct (WT) and GFP-vimentin-C328S construct (C328S) in Vim–/– mouse fibroblasts. (A) Fluorescence images showing arrangement of vimentin in WT and C328S fibroblasts. Scale bars: 50 μm. (B) Immunoblot analysis for LC3B and vimentin in transfected fibroblasts after 6 hours of serum starvation. β-Actin served as loading control. Densitometry data are representative of 3 experiments. (C) Immunofluorescence staining of pulmospheres using WT and C328 fibroblasts for LC3B (red) and green fluorescence–tagged vimentin expression after 6 hours of serum starvation. Scale bars: 100 μm. (D) Fluorescence images of IPF pulmospheres containing mEmerald-vimentin–transfected IPF fibroblasts treated with vehicle (control) or WFA. Scale bars: 250 μm. (E) Representative image of pulmospheres treated with vehicle or WFA. Graph shows calculated zone of invasion percentage from 6 IPF lung pulmospheres from controls not treated and treated with WFA. Each dot represents the mean value of calculated zone of invasion from 5 pulmospheres for each IPF subject. Data are expressed as mean ± SEM. ***P = 0.002, compared with vehicle-treated pulmospheres. (F) Transmission electron microscopy images of vehicle- and WFA-treated pulmospheres. Scale bars: 2 μm; magnified images, 500 nm. (G) Calculation formula for determination of fold change ratio for zone of invasion (ZOI) area. Median values of ZOI ≥1 represent pulmospheres nonresponsive to the treatment with WFA, and median values of ZOI <1 represent pulmospheres responsive to WFA treatment. (H) Invasiveness of pulmospheres from an individual patient not treated and treated with WFA. Box-and-whisker plots for IPF patient pulmospheres treated with WFA. Whiskers show maximum to minimum values. Lines in boxes represent median values.