Figure 3
FOXC2 is necessary and sufficient to confer EMT/CSC features, and the shift to androgen-independence/drug-resistance in PCa cells. (a) The upper panel shows morphology of LNCaP cells after FOXC2 overexpression; the lower panel shows reduction in PSA expression (in the same field) as inferred by reduced expression of GFP cloned downstream of the PSA promoter (scale bar, 100 μm). (b) qRT–PCR analyses for indicated markers. Y-axis represents fold change in HPRT-normalized mRNA expression compared with that of Vector Control cells (n=3; error bars indicate s.e.m.). (c) Immunoblotting for various markers in the indicated cell lines. (d) Quantitation of prostospheres (n=5; error bars indicate s.e.m.; **P<0.01). (e and f) Quantification of cell survival using MTS Assay in FOXC2-overexpressing LNCaP cells treated with Enzalutamide (e) or Docetaxel (f). Data are represented as absorbance (OD) at 490nm (n=3), *P<0.05, **P<0.01. (g) Morphology of indicated cell types (scale bar,100 μm). (h) qRT–PCR analyses for indicated markers in DU145 cells on FOXC2 suppression. Y-axis represents fold change in HPRT-normalized mRNA expression compared with DU145-shFF3 cells (n=3; error bars indicate s.e.m.). (i) Immunoblotting for various markers in the indicated cell types. (j) IF for AR/PSA expression in the indicated cell types (scale bar,100μm, DAPI is pseudo-colored to red). (k) Representative FACS plots for CD44 (APC) and CD24 (PE) surface marker expression analyzed in the indicated cell lines. (l) Graph shows quantification of FACS analysis shown above (n=3; error bars indicate s.e.m.; ***P<0.001). (m) Quantitation of prostospheres formed per 1000 DU145 cells on FOXC2 suppression (n=5; error bars indicate s.e.m.; **P<0.01). (n and o) Quantification of cell survival using MTS Assay in DU145-shFOXC2 cells treated with Enzalutamide (n) or Docetaxel (o). Data are represented as absorbance (OD) at 490 nm (n=3), *P<0.05.
