miR-221-5p exerts tumor suppressive function on PCa cell lines in vitro. aLeft: Relative miR-221-5p expression (2^-ΔCt) in normal prostatic epithelial Ep156T cells and AR⁺ and AR⁻ PCa cell lines. Analysis by one-way ANOVA with Tukey’s multiple comparisons test. Right: Technical replicates of at least two independent experiments for each cell line. miR-221-5p expression of AR⁺ cells was analysed by one-way ANOVA with Tukey’s multiple comparisons test. AR⁻ cell lines were compared by unpaired, two-tailed t-test. * p < 0.05, *** p < 0.001, **** p < 0.0001. Figure symbols: cross: Ep156T; circle: LNCaP; square: C4–2; triangle: VCaP; filled circle: PC-3M-Pro4luc2; filled square: DU145 cells. b miR-221-5p expression levels in PC-3M-Pro4luc2 cells upon overexpression or knock-down of miR-221-5p. LOG difference to control was calculated as LOG(2^-ΔΔCt) and analysed by unpaired, two-tailed t-test. Three technical replicates of representative experiments are shown. **** p < 0.0001 vs. scrambled, ## p < 0.01 vs. anti-scrambled. c Effect of miR-221-5p on proliferation. Images were taken 72 h post transfection with miR-221-5p and scrambled. MTS assay at 0 h, 24 h, 48 h, 72 h. Fold change compared to time point T’0 h was calculated for each time point. Data of n = 4 independent experiments are shown and were analysed by two-way ANOVA repeated measures by both factors with Sidak’s multiple comparison test. * p < 0.05, ** p < 0.01. d Effect of anti-miR-221-5p on proliferation. Images were taken at 72 h post transfection. MTS assay at 0 h, 24 h, 48 h, 72 h. Fold change compared to time point T’0 h was calculated for each time point. Data represent n = 5 technical replicates and were analysed by two-way ANOVA repeated measures with Sidak’s multiple comparison test. e Clonogenicity assay in miR-221-5p overexpressing PC-3M-Pro4luc2 cells. Dots indicate technical replicates of n = 4 independent experiments. Fold change was calculated vs. scrambled control and analysed by unpaired, two-tailed t-test. *** p < 0.001. f Clonogenicity assay in miR-221-5p knocked-down PC-3M-Pro4luc2 cells. N = 4 technical replicates are shown as fold change to control and analysed by unpaired, two-tailed t-test

Overexpression of miR-221-5p affects plasticity of PCa cells and reduces extravasation in vivo.a E-CAD/N-CAD and E-CAD/VIM ratios were calculated from the relative mRNA expression and data of n = 3 independent experiments are shown as fold change to control. Data were analysed by paired, two-tailed t-test. b Expression of E-CAD and VIM protein in PC-3M-Pro4luc2 cells 72 h post transfection with miR-221-5p and scrambled. Bands were quantified and E-CAD/VIM ratio was calculated (normalised to β-actin). The data of n = 2 independent experiments are shown as fold change to scrambled and were analysed by paired, two-tailed t-test. ** p < 0.01. c Migration of miR-221-5p overexpressing PC-3M-Pro4luc2 cells. Dots represent technical replicates of n = 3 independent experiments. Data are shown as fold change to scrambled and were analysed by unpaired, two-tailed t-test. **** p < 0.0001. d Migration of PC-3M-Pro4luc2 cells with miR-221-5p knock-down. N = 3 technical replicates are shown. Data were normalised to control and analysed by unpaired, two-tailed t-test. e Phalloidin staining in PC-3M-Pro4luc2 cells overexpressing miR-221-5p or scrambled control. F-actin signal was normalised to number of nuclei. Data was analysed by unpaired, two-tailed t-test. F-actin = green; DAPI = blue. f Extravasation of miR-221-5p and scrambled transfected PC-3M-pro4_mCherry cells at the caudal hematopoietic tissue (CHT) of zebrafish embryos 4 days post injection (dpi). Tumor burden was quantified 1dpi and 4dpi. Green = vessels; red = PC-3M-pro4_mCherry cells. Data was analysed by unpaired, two-tailed t-test. ** p < 0.01