Figure 1

DTX3L is constitutively overexpressed together with ARTD8 and ARTD9 in mPCa associated with increased IFNγ/STAT1 signaling. (A) Immunoblot analyses of untreated p53-negative, mPCa cell lines PC3 and DU145, androgen-sensitive and JAK1-negative LNCaP cell line and of the immortalized normal prostate luminal epithelial cell lines HPE and RWPE1. The HR-DLBCL-SUDHL7 cell line constitutively expressing DTX3L, ARTD9 and ARTD8[23] was used as a positive control. Whole cell extracts were separated by SDS PAGE, blotted and subsequently probed with antibodies for DTX3L, ARTD1, ARTD8 and ARTD9 pSTAT1(Y701) and tubulin. (B) Immunoblot analyses of STAT1-signaling in p53-negative mPCa cell lines PC3 and DU145 and in the androgen-sensitive and JAK1-negative LNCaP cell line treated with or without IFNγ or IFNαβ. PC3, DU145 and LNCaP cells were treated with or without IFNγ (200 U/ml) or IFNαβ (50 U/ml each) for 2 h and then whole cell extracts separated by SDS PAGE and subsequently probed with antibodies for DTX3L, ARTD9, STAT1, pSTAT1(Y701), pSTAT1(S727) and tubulin. The immunoblots are representative of at least three independent experiments. (C) Immunofluorescence microscopy analyses and sub-cellular localization of endogenous STAT1, pSTAT1-(pY701) and pSTAT1-(pS727) in PC3 cells, in presence or absence of 1000 U/ml IFNγ. Original magnification × 400. Images are representative of at least three independent experiments. (D) Immunoblot analyses of basal expression levels of IRF1 in PC3, DU145 and LNCaP cell lines. Whole cell extracts were separated by SDS PAGE and subsequently probed with antibodies for IRF1 and tubulin. The immunoblot is representative of at least three independent experiments. (E) Immunofluorescence microscopy analyses and sub-cellular localization of endogenous DTX3L, ARTD8 and ARDT9 in PC3 cells, in presence or absence of 1000 U/ml IFNγ. Original magnification × 400. Images are representative of at least three independent experiments.