p130Cas-dependent Cox-2 transcriptional expression sustains mesenchymal features of A17 cells. (A) Left panels: extracts from A17 cells expressing scramble (Ctr shRNA), p130Cas shRNAs (p130Cas shRNA) or reconstituted with human full-length (hFL) p130Cas (p130Cas shRNA + hFL p130Cas) were blotted with antibodies to p130Cas and Cyclooxygenase-2 (Cox-2). Actin was used as loading control. Middle panel: quantification of Cox-2 mRNA by qRT-PCR in cells as in (A) (**P <0.001). Right panel: luciferase activity assay in p130Cas silenced cells transfected with pGL3 vectors carrying luciferase reporter gene downstream of a short (-965, +39) or long (-3195, +39) stretch of Cox-2 promoter in presence or absence of doxycycline (*P <0.05 and **P <0.001). Negative control: pGL3 vector lacking promoter. Positive control: pGL3 vector, in which the luciferase expression is driven by SV40 promoter. (B) Left panels: Ctr and p130Cas silenced A17 cells treated with doxycycline for four days were plated on Collagen I-coated dishes for different times. Cell extracts were analyzed by blotting with antibodies to Cox-2. Actin was used as loading control. Right panel: quantification of adhesion-dependent Cox-2 mRNA by qRT-PCR (*P <0.05 and **P <0.001). (C) Upper panels: representative images of one out of three experiments performed in Ctr shRNA cells (a), p130Cas shRNA cells (b) or p130Cas shRNA cells reconstituted with hFL Cox-2 (p130Cas shRNA + hFL Cox2) (c) cultured in presence of doxycycline for four days (20X magnification). Lower panel: quantification of length/width ratio on five distinct microscope fields in each condition (**P <0.001). (D) Right panels: cell extracts were probed with antibodies to Cox-2, Snail, Slug, and Twist. Actin was used as loading control. Left panels: quantification analysis and statistic were performed on three independent experiments. (E) Left panels: representative images from A17 cells expressing scramble (Ctr shRNA) (a) or Cox-2 shRNAs (Cox-2 shRNA) (b) treated with doxycycline for four days (20X magnification). Right panels: extracts from Ctr or Cox-2 silenced A17 cells were blotted with antibodies to Cox-2, Slug, Twist, and Actin, used as loading control.