Figure 5.

Effects of anosmin-1 on cell adhesion and survival. (A) The percentage of cells adhered to the fibronectin-coated plates after 1 h at 37 °C was quantified and normalized to the nonspecific total adhesion on the poly-l-lysine-coated plate. LN229 His-KAL cells show 23% reduction (**P=0.0044) and U87MG KAL-GFP show 21% reduction (**P=0.0011) in cell adhesion, compared with the empty vector control. Experiments were performed in quintuplicate and repeated five times. Expression of the transfected anosmin-1 constructs is confirmed by anti-His or anti-GFP antibodies. (B) Effects of KAL1 knockdown on apoptosis. Caspase3/7 activity of A172 cells infected with shRNA was measured in relative light units and the average fold induction from four independent experiments is shown. P values are 0.0504 (for shRNA 673), 0.0086 (for shRNA 675), and 0.0002 (for shRNA 676). (C) Effects of KAL1 knockdown in phosphorylation status of FAK, AKT, and ERK in A172 cells. The relative ratio of phosphorylated vs total protein assessed by densitometry is shown as fold induction compared with the control shRNA, normalized to β-actin loading control. (D) Induction of PARP protein cleavage by KAL1 knockdown. The full length PARP protein is assessed by western blot in shRNA-infected A172 cells before (FBS) and after serum-starvation (SFM). The densitometry ratio is shown as fold induction compared with the control shRNA, normalized to the β-actin loading controls.