*2.6. LINC01133 Is Not a Regulator of EMT in 12Z Endometriosis Epithelial Cells*

The enrichment of differentially expressed genes associated with a mesenchymal phenotype (Figure S2, Table S6A,B) and an increased invasion of 12Z cells following *LINC01133* knockdown (Figure 3B) indicated that these cells may be converted to a more mesenchymal phenotype. To evaluate the role of *LINC01133* in epithelial to mesenchymal transition (EMT) in endometriosis, we further investigated expression of selected EMT regulatory proteins indicated to be differentially expressed in our RNA-seq data (Table S7). qRT-PCR and Western blot analysis confirmed the loss of CDH1 (E-cadherin) following *LINC01133* knockdown (Figure S3A). The levels of *CDH1* transcription correlated with the efficiency of the *LINC01133* knockdown in 12Z cells (Figure 2A, Figure S3A left panel), further supporting the involvement of *LINC01133* in its regulation. Specifically, a knockdown of *LINC01133* to about 10% of the levels of controls (*LINC01133a* siRNA, adjp < 0.0001), led to reduction of the relative levels of *CDH1* expression to 20% of the controls (adjp < 0.0001), whereas *LINC01133* knockdown to 35% (*LINC01133b* siRNA, adjp < 0.0001) led to a 30% reduction of *CDH1* transcript (*p* = 0.007) compared to controls. However, we did not see a classical EMT-associated Cadherin switch in *LINC01133* knockdown cells. The levels of *CDH2* (*N-cadherin*) transcript were downregulated to 53% of normal level in cells with high efficiency of *LINC01133* knockdown (*LINC01133a* oligo, adjp = 0.002), but were not changed in cells with a less efficient knockdown of *LINC01133* (*LINC01133b* siRNA oligo, adjp > 0.05) (Figure S3A, left panel). Further, we validated the significant downregulation of *VCAM-1* (*p* = 0.029, Figure S3B), and significant upregulation of *SOX4* (*p* = 0.02, Figure S3C) and *TGFβ2* (*p* = 0.0014, Figure S3D) in *LINC01133* knockdown cells compared to controls for both the *LINC01133a* and *LINC01133b* oligos. However, we could not validate the reduction of *KRT7* and *KRT19* (Figure S3E,F) in cells with *LINC01133* knockdown, when compared to siRNA controls. Given that we did not see a classic E-cadherin to N-cadherin switch, and that expression changes for some EMT markers could not be validated, these data suggest that *LINC01133* does not play a significant role in regulating EMT in endometriosis epithelial cells.
