Supplementary Figure 1: Characterization of the EMT induced by hnRNP E1 knockdown and validation of the splicing model in mouse.

(a) EMT induction following shRNA-mediated silencing of hnRNP E1 in NMuMG cells. Imaging of cells reveals a significant morphological change in cellular phenotype from an epithelial-like to a mesenchymal-like phenotype. EMT was validated by immunoblotting analysis using antibodies to the mesenchymal marker vimentin and the epithelial markers ZO2 and E-cadherin. Scale bar, 100 μM. (b) Quantitative RT–PCR analysis of mRNA-PNUTS, ZEB-1 and ZEB-2 expression in 24 human breast tumor samples. Relative expression levels of transcripts were calculated using the ΔCt method normalizing to GAPDH. Correlations between transcript expression levels were then evaluated using Pearson correlation coefficient test. (Linear regression, df = 24–2, a Pearson score >0.515 and P < 0.05 was considered as significant). Source data are available in Supplementary Table 2. (c) Sanger sequencing result of the lower band obtained by end-point RT–PCR in Fig. 1h. (d) Identification of an alternative splice product corresponding to the lncRNA-PNUTS in NMuMG cells. Sequence alignment of human and murine genomes indicates that exon-11 of the murine genome matches with exon 12 of the human genome. RT–PCR amplification of exon10-exon11 junction demonstrates a potential alternative splice product. The lower band was cloned and then sequenced using Sanger technology. (e) Sequencing results indicate a splicing pseudosite located 86 nucleotides downstream of the regular splicing site (Top). Schematic representation of the alternative splice region of the PNUTS variants based on Sanger sequencing results (Bottom). (f) Secondary structure of the BAT-like element located in the alternative splicing site of murine PNUTS RNA as predicted using the Mfold algorithm (ΔG = −2.10 kcal mol⁻¹). (g) Extended time course experiment using RT–PCR analysis of PNUTS gene processing following addition of TGFβ in A549. GAPDH was used as a loading control.