Fig. 2
LncRNA, NORAD, Rescues miR-346-Induced DNA Damage and is Associated with Adverse Prostate Cancer Outcome. A Western blot analysis of phospho-Ser139-γH2AX protein levels in C42 cells transfected with miR-346 (10 nM) ± pcDNA3.1-NORAD or empty plasmid for 96 h. B,C) Western blot analysis of phospho-RPA32(Ser33) protein levels in B) C42 cells transfected with miR-346 (10 nM) ± pcDNA3.1-NORAD or empty plasmid for 72 h and C) C42/shNORAD monoclone #2–12 treated with indicated doxycycline concentrations for 72 h. A-C Representative images of three independent experiments are shown, β-actin and VCL were used as a loading controls. D, E PSA relapse-free survival and distant metastasis-free survival (D) and overall survival (E) of PC patients dichotomised into NORAD low and high groups in the Walker et al. radical prostatectomy cohort (n = 322) [35] (D) and MSKCC cohort [36] (GSE21032, n = 218) (E). F Distal metastasis-free survival, PC-specific survival and overall survival in patients from six retrospective radical prostatectomy cohorts (n = 1567) with long-term clinical outcomes [37,38,39,40,41,42] accessed from the GRID database and quartiled according to NORAD Activity Score (NAS). G Correlation of NORAD expression with DNA damage response score in indicated patient tumour gene expression data sets [26,27,28,29,30,31,32,33]. H, J NORAD RNA in situ hybridisation analysis of NORAD RNA copies in matched castration-sensitive (CSPC) and metastatic castration-resistant PC (mCRPC), n = 23, LN = lymph node, LIV = liver, ST = other soft tissue. Representative images of primary tumours are shown (A - low, medium and high expression, scale bar = 100 μm, brown = NORAD RNA). * P ≤ 0.05. See also Fig. S5–7, Table S1