Fig. 11

Activation of TGF-β1 signaling and stimulation of myofibroblast differentiation by OPN in cultured lung fibroblasts exposed to MWCNTs. Cells were treated with DM, 5 μg/ml MWCNTs, or 5 μg/ml MWCNTs with TGF-β1 neutralizing antibodies, TGF-β receptor inhibitor SB525334, or OPN neutralizing antibodies for 24 h. (a) Expression and nuclear localization of p-Smad2/3 were examined by immunofluorescence (red). Blue indicates nuclear staining (scale bar: 20 μm). Cells positive for nuclear p-Smad2/3 (shown in purple color on the images from the overlapping of red and blue signals) are marked by orange arrows. (b) Expression of α-SMA was examined by immunofluorescence (green). Blue indicates nuclear staining (scale bar: 20 μm). (c) Expression and nuclear localization of p-Smad2/3 in myofibroblasts (α-SMA positive) and fibroblasts (α-SMA negative) were examined by double immunofluorescence staining of p-Smad2/3 (red) and α-SMA (green). Blue indicates nuclear staining (scale bar: 20 μm)