For the scratch assay, sh-ITGA5 and sh-NC hPSCs were seeded in a 24-well plate (6 × 104 cells per well) and allowed to become confluent. A standardized scratch was made using a 200-μl pipette tip fixed in a custom-made holder. Then, cells were washed and incubated in fresh serum free media without growth factors. Images were captured at t = 0 hours and t = 15 hours under an inverted microscope. Images were analyzed by ImageJ software to calculate the area of the scratch and represented as the percentage of wound closure compared to control cells.

The transwell migration assay was carried out in a 24-well modified Boyden chamber kit (8-μm pores, Corning Inc., Corning, NY, USA). PANC-1 tumor cells (5 × 104) were seeded in serum-free medium in the inserts with conditioned media from sh-NC or sh-ITGA5 activated either with or without TGF-β as a chemoattractant. After 16 hours of incubation, cells that migrated on the underside of the membrane were fixed in ice-cold 100% methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Migrated cells were counted in four random fields at ×100 magnification.

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