Fig. S5.

Analysis PRUNE2 and PCA3 regulation through ADAR-mediated mechanisms. (A) RT-qPCR on fractionated RNA from cytosol (C) or nucleus (N) from LNCaP cells stably expressing ADAR1-shRNA or negative control constructs. (B) Quality control of cytosolic and nuclear RNA-fractionation by agarose gel electrophoresis after RT-PCR amplification. PRUNE2 and PCA3 pre-mRNA species are detected mostly in the nucleus as well as the controls HYOU1 and SON (control nuclear mRNAs); in contrast, GAPDH mRNA (control cytosolic mRNA) is detected mostly in the cytosol. (C–E) HeLa cells stably expressing intron6-PRUNE2-GFP were transduced with either PCA3 or control constructs. Cells were analyzed for reporter GFP expression by FACS (C) and by immunoblotting with anti-GFP antibodies (D) after 24, 48, and 100 h. HeLa cells stably expressing intron6-PRUNE2-GFP, PCA3, ADAR1-shRNA, nontargeting shRNA control, or negative control (empty) lentivirus were analyzed after 48 h for the reporter GFP expression (E). (F) Human tumor cell lines stably coexpressing PCA3-luciferase (Luc) were transduced with intron6-PRUNE2-GFP or negative control expression vector. Tumor cells were lysed and luciferase activity was measured at 36 h after transduction. *P < 0.05, **P < 0.01, ***P < 0.001. (G and H) Evaluation of PRUNE2 levels in LNCaP cells stably expressing ADAR1- or ADAR2-shRNA, and control lentivirus. Quality control of two individual ADAR1- and ADAR2-shRNA constructs (G). Whole cell extracts from LNCaP cells expressing ADAR1- and ADAR2-shRNA constructs were probed with antibodies against PRUNE2 and tubulin (H).