Figure 4

VB6 chromobody visualizes distribution and reorganization of vimentin upon stimulation and knockdown of vimentin.

(a,b) A549_VB6-CB cells were left untreated (−TGF-β) or stimulated with TGF-β (5 ng/ml) for 72 h. Cells were either subjected to immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (a) or to microscopic analysis (b). (c,d) Knockdown studies of vimentin in A549_VB6-CB cells using three vimentin-specific siRNAs (siVIM1-3) and two control siRNAs (siCTR1-2), followed by immunoblot analysis with an α-VIM-IgG and anti-GAPDH antibody (c) or microscopic analysis (b,d). Shown are representative images from three independent experiments. Scale bars: 20 μm. (e) Time-lapse microscopy of A549_VB6-CB cells. Cells were stimulated with TGF-β (5 ng/ml) for 48 h. Subsequently, TGF-β was removed and cells were cultivated for additional 45 h. Images were taken in 3 h intervals, shown are representative images at indicated time points. Scale bar: 20 μm.