Matrigel coated chamber

Cell migration assay was performed using 8 μm transwell chambers (Falcon, USA). Cell invasion assay was performed using Matrigel-coated chambers (BD Biosciences, USA). After 24 h incubation at 37 °C, the cells on the upper surface of the membrane were removed, the membranes were stained with 0.1% crystal violet for 15 min, and the cells on the lower side were then counted under a microscope.

PDCs or TNBC cell lines were seeded at a density of 3 × 10⁵ onto noncoated or Matrigel-coated chambers (BD Bioscience) with KYA1797K or DMSO. Cells were allowed to invade for 24 h. After clearing cells on the inner surface of the chamber, the cells on the outer surface were fixed in 4% paraformaldehyde (PFA) for 15 min and stained with crystal violet for 20 min. The chambers were dipped in distilled water to remove excess staining and allowed to dry. Representative images were captured by microscopy (TE-2000U, Nikon). Mean ± SD are reported based on three biological replicates.

Transwell^® invasion experiments were performed with 24-well plates with Matrigel™-coated chambers (8 μm pore size) from BD Biosciences. Briefly, cells were allowed to grow to subconfluency (~80%) and serum starved for 24 h. Following detachment with trypsin, cells were washed with PBS, resuspended in serum-free medium and 2×10⁴ cells were added to the upper chamber. Complete medium was added to the bottom wells of the chambers. After 24 h, the cells that had not migrated were removed from the upper face of the filters using cotton swabs, and the cells that had migrated were fixed and stained with crystal violet solution. Cell migration assays were performed similarly, however, without Matrigel™ coating.

Cell migration was assessed by measuring the movement of cells into a scraped, acellular area that was prepared as described previously.^{48 (link)} The spread extent of wound closure was observed after 0 and 48 h, respectively. Migration was quantified by counting the total number of cells that migrated toward the original wound field. For the invasion assay, Matrigel-coated chambers (BD Biosciences, San José, CA, USA) containing 8-μm pores were used. A total of 2 × 10⁵ concentration cells were seeded into the upper chambers (coated in Matrigel) in serum-free medium. The lower chamber of the Transwell was filled with culture media containing 10% FBS as a chemo-attractant. After the chambers were incubated at 37 °C for 48 h, non-invaded cells on the top of the Transwell were scraped off with a cotton swab. Successfully translocated cells were fixed with 10% formalin. Then, they were stained with 0.1% crystal violet for 30 min and counted under a light microscope. All experiments were performed in triplicate.