*4.8. Migration Assays*

Boyden Chamber Migration Assays (8.0 μm pore size, 353097, Falcon, Corning, Amsterdam, The Netherlands) were performed to assess cell motility and migration. The cells were plated 48 h post-transfection in the upper chamber of the migration inserts with starved RPMI medium (0% FCS), whereas the lower chamber was filled with standard medium containing 10% FCS for chemotactic attraction. The experiment was stopped after 48 h of incubation, the cells being fixed with 4% formaldehyde and colored with hematoxylin. Membranes were scanned, and the cells were counted automatically by nucleus detection using the QuPath software (v0.2.0-m6) [7,32].

#### *4.9. Statistical Analysis*

Microsoft Excel (v16), SPPS (v25), and GraphPad Prism (v8) were used for statistical analyses and visualization of the data. The nonparametric Mann–Whitney U or Kruskal–Wallis test were used for group comparisons. Pearson´s correlation coefficients were calculated. Survival analyses were performed using Kaplan Meier estimate curves and log-rank tests. Thus, multivariate Cox regression analyses were performed after co-adjustment of the TNM stage (the only *n* = 3 M1 in PCa TCGA were excluded; in PCa TMA no cM1 cases) and age to evaluate an independent and additive prognostic value on patients' progression-free survival.
