Fig. 5: edAZIN1 fails to induce an aggressive phenotype in Myosin9 knockout cells.

Using CRISPR/Cas9 gene editing and specific sgRNAs, Myosin 9 protein expression was successfully knocked out in PC3 (a) and DU145 (c) cells. Myosin 9-knockout PC3 (b) and DU145 (d) cells were transfected with 2,5 µg of pcDNA3.1-Clover-edited-AZIN1 for 24 h and analyzed by confocal microscopy. The effect of overexpressing the edAZIN1 allele in Myosin 9 knockout PC3 and DU145 cells with regard to cell proliferation (e and f), invasion into the extracellular matrix (g and h), and colony formation in soft agar (i). Proliferation was measured by MTT reduction. Invasion through Matrigel supported by a Transwell membrane (8 μm pores) was assessed after methanol fixation and toluene blue staining. Soft agar colony formation was assessed by incubation for 19–21 days, fixation, and crystal violet staining. The morphology of PC3 (j) and DU14 (k) cells is shown, and representative images acquired by light microscopy (lens: original magnification, 10X) are presented. The bars indicate the mean (±S.D.) from three independent experiments. * significant difference compared to empty vector (P < 0.05), ** significant difference compared to empty vector (P < 0.01) and *** significant difference compared to empty vector (P < 0.001). Myosin 9 knockout PC3 (l) and DU145 (m) cell populations were analyzed by western blotting with the indicated antibodies.