Figure 3
Effect of PSAP down-modulation on β 1A -integrin expression and PCa cell adhesion to FN and LN. (A) PSAP down-modulation reduces β1-integrin expression. Total RNA was extracted for RT-PCR with primers specific for integrin-β1A, -β1B, and -β1C, and GAPDH. Cell lysates were analyzed by western blotting with antibodies against β1-integrin and its isoforms β1A, β1B, and β1C and GAPDH. (B) Transient down modulation of β1-integrin expression. Previously established control stable clones of PC-3 and DU-145 cells were transiently transfected with β1 integrin- or scrambled-siRNA oligos. After 48 h, cell lysates were analyzed for integrin β1 and β1A expression by western blotting. (C) Inhibition of PCa cell adhesion by transient transfection with integrin β1-siRNA. After siRNA transfection, cells were subjected to adhesion assay on FN- or LN-coated 96-well plates as described in "Materials and Methods". (D) β1A-integrin stability. The steady-state β1A protein levels were evaluated by treating cells with cycloheximide (12.5 μg/ml) for up to 72 h and immunoblotting with anti-β1A antibody. (E) The half-live of β1A-integrin was evaluated by densitometric analysis of immunoblotting bands using the Quantity One software and the β1A protein levels were calculated as percentage of non-treatment values after normalization using GAPDH for loading control. (F) Effect of inhibition of lysosomal proteolysis on β1A-integrin expression. Cells were incubated in the presence or absence of the lysosomal proteolysis inhibitor, NH4Cl (50 mM) for up to 24 h. Cell lysates were analyzed for β1A protein expression by immunoblotting. The β1A-integrin degradation curve was calculated as described above. Columns, mean of three independent samples run together; bars, ± SEM, p < 0.0001, Two-sample t-tests with Satterthwaite corrections were used to compare β1-siRNA versus scrambled siRNA oligos transfected cells following adhesion to FN or LN. ANOVA was used to examine the significance of the data (p < 0.05) among different cycloheximide treatment periods in PSAP-KD versus control clones. Similar results were obtained from three independent experiments.