Figure 3

Loss of β1 integrin expression stimulates cell adhesion and spreading to rTGFBI in ovarian cancer cells. A, SKOV3 cells infected with Lentivirus expressing shRNA against β1 integrin. Western blot analysis of RIPA soluble lysates utilizing antibodies against β1 integrin or alpha-tubulin. Bright-field (a,c) and confocal immunofluorescence microscopy (b,d) images of control non-target shRNA (a,b) or β1 integrin shRNA (c,d) treated cells following adhesion to rTGFBI. Rhodamine-phalloidin was utilized to visualize the actin cytoskeleton. Scale bar = 40 μm (confocal), scale bar = 50 μm (bright-field). B, SKOV3 cells were either control non-target shRNA or β1 integrin shRNA treated followed by incubation on either fibronectin, rTGFBI, or rPeriostin coated tissue culture plastic for 30 minutes. Results of three independent experiments were normalized to poly-L-lysine and represented as percent of non-target control shRNA on each matrix protein. Significance of *p < 0.05 and **p < 0.01 is compared to control shRNA. C, SKOV3 cells infected with Lentivirus expressing shRNA against β3 integrin. Cells expressing either non-target shRNA or β3 integrin shRNA were replated on fibronectin, rTGFBI, or rPOSTN coated wells and allowed to adhere for 30 minutes. Results of three independent experiments were normalized to poly-L-lysine and represented as percent of non-target control shRNA on each matrix. Significance of *p < 0.05 and ***p < 0.001 is compared to control shRNA. D, PEO1 cells expressing either non-target shRNA or β1 integrin shRNA were replated on fibronectin, rTGFBI, or rPOSTN coated wells and allowed to adhere for 1 hour. Results of three independent experiments were normalized to poly-L-lysine and represented as percent of non-target control shRNA on each matrix. Significance of *p < 0.05, **p < 0.01, and ***p < 0.001 is compared to control shRNA. E, SKOV3 cells following siRNA transfection against β1 integrin or non-target control were processed for live cell immunostaining against the αvβ3 integrin heterodimer using the LM609 antibody (green). Hoechst stain was utilized to visualize the nuclei (blue). Scale bar 40 μm. Quantitation of live cell immunostained avβ3 integrin heterodimers was achieved using ImageJ software. All experiments were performed in duplicate and analysis was performed on greater than 100 cells/experiment. Results are represented as αvβ3 integrin cell surface fluorescence intensity compared to control siRNA cells.