(A) Western blot showing efficient knockdown of PODXL in SW1990 pancreatic cancer cells using shRNA lipofectamine transfection followed by stable clonal selection. (B) Representative time-lapse micrographs of scramble control and PODXL-KD SW1990 cells migrating on 2D collagen I-coated glass coverslips. The images are spaced at 2 h intervals. (C) Average mean squared displacement (MSD) over time of migrating scramble control and PODXL-KD SW1990 cells. Data represent the mean±S.E.M from 3 independent experiments. (D) Superimposed images of individual trajectories of scramble control and PODXL-KD SW1990 cells migrating on 2D collagen I-coated surfaces. (E) Representative time-lapse micrographs of scramble control (top row) and PODXL-KD (bottom row) SW1990 cells migrating inside 6 μm-wide, confined microchannels (left panels) and 50 μm-wide, unconfined microchannels (right panels). The images are spaced at 2 h intervals. Arrowheads indicate the leading edge of a migrating cell. (F) Migration velocity (left panel), speed (middle panel) and persistence (right panel) of scramble control and PODXL-KD SW1990 cells in PDMS-based microchannels of 10 μm in height, 200 μm in length, and either 6, 10, 20 or 50 μm in width. Data represent the mean±S.E.M from at least 3 independent experiments. * represents p<0.05 between scramble control and PODXL-KD cells.