## 2 Results
### Upregulation of Inc-Nr6a1 and Embedded miR-181a2 and miR-181b2 during EMT
**(C,D)** Effect of MAP kinase inhibitor U0126 and AKT inhibitor GSK690693, respectively, on Inc-_Nr6a1_ isoforms upregulation. NMuMG cells were pre-treated with 10 \(\upmu\)M inhibitor for 1 h prior to addition of TGF-\(\beta\) for 2 h where indicated. Levels of Inc-_Nr6a1-1_ (top) and Inc-_Nr6a1-2_ (bottom) were measured by RT-qPCR analysis. Experiments were performed in triplicate samples; error bars represent S.D. **(B)** Effect of p38 kinase inhibitor, SB203580, on Inc-_Nr6a1_ isoforms upregulation. Cells were pre-treated with 10 \(\upmu\)M  
performed in triplicate samples; error bars represent S.D. (**E**) Representative immunoblot (top) and densitometry analysis (bottom) for SMAD4, p38 kinase (phosphorylated p85 ribosomal kinase, p85), MAP kinase (phosphorylated ERK1/2, PERK1/2) and AKT kinase (AKT phosphorylated substrate, AKT Sub) are shown. \(\beta\)-actin was used for protein-loading normalization in SMAD4, p38 kinase and AKT kinase immunoblotting. Total ERK1/2 were used as the protein-loading control in the ERK1/2 immunoblot. (**F**) Requirement of de novo protein synthesis for Inc-_Nr6a1_ isoforms upregulation by TGF-\(\beta\). NMuMG cells pre-treated with 5 uM of cycloheximide (CHX) for 1 h were stimulated with TGF-\(\beta\) for 2 or 24 h. Expression levels of Inc-_Nr6a1_ isoforms and SIP1 were measured by RT-qPCR analysis. Experiments were performed in triplicate samples; error bars represent S.D. ***\(p<0.001\), **\(p<0.01\), ns, not significant by two-tailed Student's \(t\)-test. Signaling pathways involved in Inc-_Nr6a1_ isoforms induction by TGF-\(\beta\) were investigated. Smad4 is a central mediator of the Smad-dependent canonical pathway. Two different Dicer-substrate short interfering RNAs were used for silencing Smad4 and the knockdown efficiencies were tested by Western blotting. Upregulation of Inc-_Nr6a1_ isoforms by TGF-\(\beta\) was not affected in Smad4-suppressed cells, indicating TGF-\(\beta\)-Smad signaling independency of TGF-\(\beta\) response (Figure 2A). TGF-\(\beta\) non-canonical pathways, extracellular signal-regulated kinases (ERKs), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (AKT) are activated very rapidly in TGF-\(\beta\)-treated NMuMG cells [15]. Blocking of p38 MAP kinase and AKT signaling pathways had no effect on TGF-\(\beta\)-upregulation of Inc-_Nr6a1-1_ (Figure 2B,D), whereas blocking the MEK-ERK1/2 signaling pathway inhibited its expression (Figure 2C). Remarkably, Inc-_Nr6a1-2_ expression was specifically abolished by GSK6906693, an inhibitor of AKT (Figure 2D). Taken together these results indicate that each isoform is regulated by a different signaling pathway. The early upregulation of Inc-_Nr6a1-1/2_ isoforms suggested an active role in the start of the EMT program. To determine whether they were direct targets of ERK and AKT signaling pathways, expression levels of Inc-_Nr6a1-1/2_ isoforms were analyzed in cells treated with TGF-\(\beta\) in the presence or absence of cycloheximide, an inhibitor of protein synthesis. RT-qPCR results showed upregulation of both isoforms in the presence of cycloheximide, whereas upregulation of an indirect target control, SIP1, was blocked (Figure 2E). These data suggest that Inc-_Nr6a1-1_ and Inc-_Nr6a1-2_ isoforms are direct targets of ERK1/2 and AKT pathways, respectively.