Figure 7

Increased MMP-9 expression and CREB-1 phosphorylation in InsR silenced cells. (A) Cell lysates from SC- and sh-InsR-transfected cells were subject to Western blotting using an antibody against phosphorylated (P-CREB-1) or total CREB-1 (T-CREB-1). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. *, P<0.01 vs. SC transfected cells. (B) Scrambled oligo (SC)- and sh-InsR-transfected cells were co-transfected with pUSEamp⁽⁺⁾ empty vector or a dominant negative (DN)-Akt vector. Cell lysates were subject to Western blotting for CREB-1 as described in (A). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. *, P<0.01(lower panel). (C) Cells were stably co-transfected with plasmids as indicated and lysates were subject for Western blotting for MMP-9. pRS, a control plasmid for InsR shRNA; pUSE, pUSEamp⁽⁺⁾, a control plasmid for DN-Akt; pGeneClip, a control plasmid for CREB-1 shRNA (upper panel). An image of gel stained after transfer was shown as a loading monitor (middle panel). The bar graph shows the densitometric scanning results (means ± SEM) from four independent experiments. *, P<0.01. (D) Scrambled oligo (SC)- and sh-InsR-transfected cells were incubated with doxycycline (10μg/mL, 72 hours) and cell lysates were subject to Western blotting for FN. An image of gel stained after transfer was shown as a loading monitor (middle panel). The bar graph shows the densitometric scanning results (sh-InsR/SC, means ± SEM) from four independents experiments. *, P<0.01.