## 2 Results
### Lnc-_Nr6a1_ Depletion Reduces Cell Adhesion and Alters Gene Expression
We found 44% and 27% of upregulated and downregulated genes, respectively, by TGF-\(\beta\) were common in both types of cells; therefore, their regulation was independent of the presence or absence of _Lnc-Nr6a1_. These genes were enriched in those involved in EMT, phosphatidylinositol processing, DNA replication checkpoint and chromatin silencing (Figure 5A,B). This result indicates that 56% and 73% of genes induced or inhibited, respectively, by TGF-\(\beta\) are dependent on the presence of _Lnc-Nr6a1_ and slightly enriched in genes involved in histone modification and ribosomal RNA maturation. Likewise, _Lnc-Nr6a1_ depletion caused induction or inhibition of new genes by TGF-\(\beta\) (39% and 55%, respectively) enriched mainly in meiotic chromosome condensation, DNA replication initiation and negative regulation of chromatin silencing. Together, these data indicate that _Lnc-Nr6a1_ depletion affects differential gene expression mediated by TGF-\(\beta\); however, expression of genes involved in EMT was independent on the presence of _Lnc-Nr6a1_. Forced expression of Inc-Nr6a1_ isoforms neither revealed apparent morphological changes in cells nor significant changes in expression levels of the EMT markers. Furthermore, neither cell cycle nor proliferation were affected in isoform-overexpressing cells (Figure S8). However, wound-healing assays revealed that cells overexpressing either of the two isoforms migrated much faster than control cells, but the effects were not additive. Notably, cell migration was not affected by _Lnc-Nr6a1_ depletion (Figure 5C-E, Video S1). Figure 3: Deletion of a _Luc-Nr6a1_ fragment in NMuMG cells and expression profile. (**A**) Scheme showing the 600 bp deleted fragment of _Luc-Nr6a1_ deletion in NMuMG cells using CRISPR-Cas9 technology. Sites of cleavage by Cas9 in the promoter and first exon regions are indicated; primers (green arrows) for clones analysis are shown (**B**) Representative gel identifying non-deletion (control, clones 1, 7 and 8) monoallelic (clone 2, 3, 4, 5 and 6) and biallelic clones (clone 9). (**C**) Expression levels of _Inc-Nr6a1_ isoforms in different clones were measured by RT-qPCR analysis. (**D**) Expression of miR-181a/b species in control and _Luc-Nr6a1_-depleted cells after 24 h of TGF-ß treatment or untreated. Experiments were repeated twice in triplicate samples; error bars represent S.D. *** \(p<0.001\), *** \(p<0.01\), *** \(p<0.05\), ns, not significant by two-tailed Student’s \(t\)-test. Y-axis of last panel (down right) is labeled 181a-5p because the isoforms miR-181a1-5p and miR-181a2-5p are identical. (**E**) Volcano plot for differentially expressed genes in _Luc-Nr6a1_-depleted cells versus control cells. Genes upregulated with more than a 1.5-fold change with a \(p\)-value \(<0.05\) are depicted in red boxes and those downregulated with identical fold change and \(p\)-value are in green boxes. (**F**) Gene ontology enrichment analysis of downregulated genes in _Luc-Nr6a1_-depleted cells. Figure 4: _Inc-Nr6a1_ depletion affects cell adhesion. Scanned 60-mm Petri dishes with fixed colonies subjected to crystal violet staining assays, **(A,B)** control cells and _Inc-Nr6a1_-depleted cells, **(C)**_Inc-Nr6a1_-depleted cells and _Inc-Nr6a1_-depleted cells overexpressing _Inc-Nr6a1_-1 isoform. Cells were plated on dishes and incubated for indicated times. Stained adherent cells were solubilized with 1% SDS at room temperature for 30 min and quantified at OD 590 nm. Experiments were performed in triplicate, and data are expressed as the mean \(\pm\) S.D. from three independent experiments. **(D,E)** Flow cytometric analysis of control, _Inc-Nr6a1_-depleted and _Luc-Nr6a1_-depleted cells, overexpressing _Inc-Nr6a1_-1_ calcium-labeled cells. Labeled cells were added to a confluent monolayer of unstained NMuMG cells incubated for 2 h and adherent cells quantified by flow cytometry analysis. The bar graph shows mean \(\pm\) S.D. of six independent experiments, \(***\)\(p\)\(<\) 0.001, \(**\)\(p\)\(<\) 0.01 via two-tailed Student’s \(t\)-test.