Stable knockdown of Id4 by retroviral shRNA in LNCaP cells (retroviral vectors A and C) and stable over-expression of hId4 in Du145 cells. A. Real time quantitative polymerase chain reaction for Id4 expression in LNCaP (NS, non-specific) following transfection with Id4 shRNA vectors A and C and non-silencing shRNA (NS) (***: P < 0.001). B. Western blot analysis of Id4 expression in LNCaP cells with non-specific shRNA (NS) and Id4 specific shRNA (-Id4, vector A). C: Immuno-cytochemical analysis of stable knockdown of Id4 expression in LNCaP cells (LNCaP-Id4, vector A) as compared to cells with non-specific shRNA (LNCaP + NS). The red staining indicates Id4 expression (DyLight 594). Id4 expression in DU145 cells stably transfected with Id4 expression vector (DU145 + Id4) as compared to DU145 cells transfected with empty vector (DU145 + NS). The green staining represents Id4 (DyLight 488). DAPI was used to stain the nuclei (blue) in both LNCaP and DU145 cells. Representative images are shown.