ASB2-induced degradation of FLNs inhibits cell spreading on fibronectin. (A,B) NIH3T3 cells were transfected with GFP, GFP-ASB2wt, GFP-ASB2LA, or GFP–ASB2-Δ expression vectors for 24 hours, trypsinized, and serum arrested for 1 hour in suspension. Cells were plated on fibronectin-coated coverslips and fixed after 45 minutes. (A) Cells were stained with anti-FLNa antibodies as indicated. Scale bar represents 20 μm. (B) Percentages of nonspread (▩) and spread (■) cells are plotted as mean plus or minus SEM from 3 independent experiments. (C) FLNa and FLNb double knockdown in HT1080. (D) Spreading quantification. HT1080 WT or HT1080 FLNabKD cells were replated on fibronectin-coated coverslips and fixed after 40 minutes. Cell areas of at least 100 cells were measured and plotted as mean plus or minus SEM. (E) HT1080 WT were transfected with GFP-ASB2wt and 24 hours later replated on fibronectin-coated coverslips, fixed after 40 minutes, and stained for FLNa. Cell areas were measured and cells expressing GFP-ASB2wt (outlined in the image) were compared with untransfected cells on the same coverslip. Scale bar represents 20 μm. The plot shows the mean area plus or minus SEM.