Figure 6

HO-1 mediates cell adhesion via PXDN in 607B melanoma cells. (A) Western blot analysis of ctrl (MSCV) or cells transduced with a HO-1 cDNA (MSCV-HO1) demonstrates efficient HO-1 overexpression in 607B melanoma cells. β-actin indicates equal loading. A representative example is shown. (B) Control-infected (MSCV) or HO-1 transduced (MSCV-HO-1) 607B cells were seeded on Fibronectin (FN), Laminin (Ln) or tissue culture plastic and assayed for adhesion as described in Materials&Methods. Note that HO-1 overexpressing 607B cells (MSCV-HO1) became more adherent than MSCV cells (*P < 0.05 vs MSCV cells). (C) PXDN mRNA levels were measured by qRT-PCR using the RNA samples isolated from control (MSCV) or HO-1 overexpressing (MSCV-HO1) 607B cells. The expression values were normalized relative to Arp. The levels of PXDN mRNA in MSCV and MSCV-HO1 cells are shown in percentage relative to MSCV cells (set to 100%). Bars represent mean (+/- SEM) of three independent experiments. (D) PXDN mRNA knocked down in 607B MSCV-HO1 cells after transient transfection with a control (siCtrl) or PXDN-specific (siPXDN) siRNA, as determined by real-time PCR. (E) Effect of PXDN-knockdown on cell adhesion to fibronectin or laminin in HO-1 overexpressing 607B cells. For comparison, OD-values of siCtrl infected cells were arbitrarily set to 100% in each experiment. Note that PXDN-silenced cells (siPXDN), but not siCtrl-treated (siCtrl) cells became less adherent following PXDN-knockdown (*P < 0.05 vs siCtrl-treated MSCV-HO1 cells).