Figure 6.

Decreased pro-invasive ability, matrix contraction capacity, and α-SMA protein levels in αv-silenced TIF fibroblasts. (A) Organotypic assay conducted with 5 × 10⁴ TIF fibroblasts, transfected with siRNA CTRL or si-αv, combined with a collagen I solution. 1 × 10⁵ HT1080-GFP cells were seeded on top of these matrices, incubated for 2 days (T0) and let to invade for 3 or 6 days. Matrices were processed as described in the legend to Figure 3. Scale bar 50 µm. (B) The number of invading HT1080-GFP cells in (A) was quantified, normalized to T0 and reported as percentage of si-CTRL fibroblasts after 3 days, considered as 100%. (C) 5 × 10⁴ TIF fibroblasts, transfected with si-CTRL or si-αv, were grown for 48 h in DMEM-10% FBS and serum-starved for 24 h. CM were collected and employed as chemoattractants for HT1080-GFP in Boyden chamber assays as described in the legend of Figure 3D. (D) Contraction assay carried out with 5 × 10⁴ TIF fibroblasts transfected with siRNA CTRL or siRNA to integrin αv and, after 24 h, mixed with the collagen I solution. The reduction of matrix diameter was monitored for 3 days. The area of matrices with TIF fibroblasts transfected with si-CTRL at day 2 was taken as 100%. (E) Western blot of total lysates from fibroblasts transfected with siRNA CTRL or siRNA to αv integrin were collected and subjected to quantitation of α-SMA and αv protein levels using GAPDH as a reference. The whole blot image can be found in Figure S8. (F) 2 × 10⁴ fibroblasts were seeded in DMEM-10% FBS on cover slips in 6 well plates for 24 h and silenced for integrin αv expression by RNA interference. After 72 h, cells were fixed and stained with DAPI or anti-α-SMA antibody by immunofluorescence (20× magnification and 63× in the inset). Scale bar 50 µm. The MFI following staining with anti-α-SMA antibody was quantified and reported as indicated in the legend to Figure 5D. * p < 0.05; ** p < 0.005; *** p < 0.001, Student’s t-test.