Supplementary Figure 3: Characterization of ARLNC1 and its expression.

a) Relative expression of ARLNC1 (FPKM) across 14 prostate cancer cell lines. (b) qPCR analysis of ARLNC1 expression in nine prostate cancer cell lines. Expression levels of several known prostate cancer-associated lncRNAs are also shown. Mean ± s.e.m. are graphed, n = 3. (c) Left: Representative image of ARLNC1 gene structure in AR-positive prostate cancer cells, generated from RACE analysis. Annotations of ARLNC1 in Ensembl and the Encyclopedia of DNA Elements (ENCODE) are also shown. Inset: Expression of ARLNC1 transcripts in MDA-PCa-2b cells, validated by Northern blot. ARLNC1-negative DU145 cells serve as negative control. Primer sequences for Northern blotting probe are listed in Supplementary Table 3. Right: 5’ RACE and 3’ RACE results in MDA-PCa-2b cells and LNCaP cells. Experiments were repeated 3 times with similar results. (d) smFISH images depicting localization of ARLNC1 transcripts in a panel of prostate cancer cell lines. Representative pseudocolored images of MDA-PCa-2b, LNCaP, VCaP, 22Rv1, PC3, RWPE, and DU145 cells probed for ARLNC1 (gray) or GAPDH (gray, control). Nucleus is stained with DAPI (blue). Scale bar, 5 µm; n = 3 independent experiments for each cell line. (e) Scatter plot representing the average number of ARLNC1 transcripts per cell in a panel of prostate cancer cell lines, including MDA-PCa-2b, LNCaP, VCaP, 22Rv1, PC3, RWPE, and DU145. Black line and whiskers depict the mean and s.e.m., respectively (n = 50 cells for each cell line aggregated from 3 independent experiments). (f) Representative gray-scale images of MDA-PCa-2b cells stained for DAPI (nucleus, blue) and ARLNC1, AR or GAPDH transcripts (smFISH, gray). Scale bar, 10 µm. Quantification of the number of molecules per cell and the nucleo-cytoplasmic distribution of each transcript is also represented (n = 60 cells for each sample aggregated from 3 independent experiments). Center line represents mean and error bars represent s.e.m. (g) Percentage of nuclear/cytoplasmic RNA levels of ARLNC1, ACTB, and U1, measured by qRT–PCR after subcellular fractionation of MDA-PCa-2b and LNCaP cells. U1 serves as a positive control for nuclear gene expression, while ACTB RNA serves as a positive control for cytoplasmic gene expression. The graphs show mean ± s.e.m., n = 3 independent experiments for each cell line.