Figure 3.

Roles of podocalyxin‐like protein (PODXL) in the motility and invasiveness of pancreatic ductal adenocarcinoma cells. (a) RNA oligonucleotides were transiently transfected into S2‐013 and PANC‐1 cells; the siRNAs targeted PODXL (siPODXL) and the negative control was a scrambled RNA (Scr). Western blotting was carried out using anti‐PODXL antibody. (b,c) Oligonucleotides targeting PODXL or Scr were transiently transfected into S2‐013 or PANC‐1 cells. Motility (b) and two‐chamber invasion assays (c) were undertaken. Migrating cells in four fields per group were scored. Data are derived from three independent experiments. Columns, mean; bars, SD. *P < 0.001 compared with Scr‐transfected control (Student's t‐test). (d) Confocal immunofluorescence microscopic images (right panels). A myc‐tagged PODXL‐rescue construct was transfected into PODXL siRNA‐transfected S2‐013 cells. Forty‐eight hours later, cells were incubated on fibronectin. Cells were stained with anti‐myc antibody (green). Actin filaments were labeled by phalloidin (red). Blue, DAPI staining. Bar = 10 μm. Western blots probed with anti‐myc and anti‐PODXL antibodies are shown in left panels. (e) Mock control vector or myc‐tagged PODXL‐rescue construct were transiently transfected into scrambled control siRNA‐transfected and PODXL siRNA‐transfected S2‐013 cells; 48 h later, motility (left panel) and two‐chamber invasion assays (right panel) were carried out. Migrating cells in four fields per group were counted. Data are derived from three independent experiments. Columns, mean; bars, SD. *P < 0.001, **P < 0.005 compared with corresponding PODXL siRNA‐transfected S2‐013 cells that were transfected with mock vector (Student's t‐test). IB, immunoblot.