Supplementary Figure 2: The predicted lncRNA-PNUTS does not encode protein, is both nuclear and cytoplasmic and colocalizes with miRNA-205.

(a) In silico prediction of PNUTS alternative splicing sites. Lower case: intronic sequences; Upper case: exonic sequences; Bold characters: natural acceptor splice site; Bold italicized characters: newly identified pseudosite. (b) Western-blot (top) analysis of PNUTS protein expression upon PNUTS predicted-lncRNA transient overexpression (3 days) or TGFβ treatment (1 day) in A549, MCF7 and CaCO2 cell lines. RT–PCR analysis (bottom) was used to monitor the PNUTS predicted-lncRNA overexpression. ^∗indicates the PNUTS protein band. The fact that the lncRNA does not appear upregulated in this experiment is due to the time point assayed (24 h TGFβ treatment) (see Supplementary Fig. 1g). (c) Fluorescence in situ hybridization analysis (FISH) of the endogenous form of lncRNA-PNUTS, mRNA-PNUTS and miR-205 was performed using selective probes labeled with ATTO dyes. Images at 300× magnifications, obtained by using confocal microscopy. Scale bar, 10 μM.