Transwell migration and invasion assay

PFKP siRNA-transfected or L-lactate-treated breast cancer cells were washed and detached with Versene (Gibco^®, NY, USA) and re-suspended as single-cell suspensions in low-serum DMEM (supplemented with 1% FBS). The cell invasion assays were performed in cell culture inserts (PET membrane with 8.0 μm pore size; Ref# 353097; Corning Incorporated, NC, USA). The cells were counted using an automated cell counter (Countess™, Invitrogen). A total of 25,000 cells in low-serum DMEM was added to the upper chamber [for rWNT5A treatment, 0.4 μg/ml rWNT5A was added to the medium], and the lower chamber was filled with 0.7 ml of DMEM supplemented with 10% FBS. The cells were allowed to invade for 24 h at 37°C in a humidified incubator with 5% CO₂. After incubation, the cells in the insert were fixed with 4% paraformaldehyde for 10 min at RT. The non-migratory cells on the upper side of the insert were removed with a cotton-tipped applicator and were further processed as published by Linnskog et al. [57]. Transwell invasion assays for PFKP siRNA-treated MDA-MB-231 cells were performed in a similar manner using BD Matrigel™ invasion chambers (MA, USA) in which 50,000 cells were added to the upper chamber. Statistical analyses were performed by taking those cells into account that have migrated or invaded to the other side of the membrane (outer side). Furthermore, it was checked that the incubation time (24 h) used in these experiments did not allow cells to detach from the membrane into the lower chamber.