EGFR knockdown reduces the sphere-forming capacity and self-renewal activity of DU145 PCSCs.

<p>A) DU145 monolayer cells were infected with EGFR shRNA lentiviral constructs and selected with puromycin in order to generate stable cell lines. Western blot analysis of various EGFR knockdown (shEGFR) cells was carried out to evaluate the EGFR knockdown efficiency relative to the non-specific shRNA control (shCTRL) cell line. B) Stable EGFR knockdown cells form fewer primary (1°) spheres compared to shCTRL cells. Cells were seeded at a density of 2.5×10³ cells/well (0.5 ml/well; three replicates per treatment). Sphere numbers are displayed as mean ± S.E.M. of three independent experiments (*<i>p</i><0.05 in comparison to the respective -EGF media condition for each cell line; two-tailed independent Student’s <i>t</i>-test). C) Efficient EGFR knockdown in established DU145 spheres can also be achieved. Cells from EGF-free (-EGF) spheres were infected with shCTRL and shEGFR2 lentivirus. Loss of EGFR protein expression significantly reduces the D) number of DU145 spheres grown under EGF-free media conditions. EGFR knockdown (shEGFR2) and shCTRL sphere cells were seeded at a density of 1×10³ cells/well (three replicates per cell line). The number of spheres that formed was counted 12 days post-seeding. Sphere number is displayed as mean ± S.E.M. of three independent experiments (**<i>p</i><0.01; two-tailed independent Student’s <i>t</i>-test). E) EGFR knockdown in DU145 sphere cells significantly reduced their in vitro anchorage-independent growth. The total number of colonies in 5 random fields (at 25X magnification) was counted and represented as mean ± S.E.M. of three independent experiments (**<i>p</i><0.01; two-tailed independent Student’s <i>t</i>-test). Representative phase contrast images of colonies are shown at 50X magnification (inset). Scale bar is equal to 100 µm.