*2.7. Active Cytoskeleton Remodeling in 12Z Cells Following LINC01133 Knockdown*

*LINC01133* knockdown led to the deregulation of genes involved in cell adhesion and EMT, and was associated with changes in the cellular phenotype of 12Z knockdown cells, which appeared to have a more flattened, larger phenotype with an increased number of actin stress fibers (Figure 5A). This was supported by analysis of cell area and fluorescence intensity in *LINC01133* knockdown cells compared to controls. We confirmed that the cross-sectional cellular area of *LINC01133* knockdown cells was greater, with knockdown cells 1.7 fold the size of control siRNA treated cells (adjp = 0.006, Figure S4A). Further, analysis of Phalloidin fluorescence intensity showed that *LINC01133* knockdown cells had 4.5-fold higher corrected total cell fluorescence (CTCF) than cells transfected with control siRNA oligo (*p* < 0.0001) (Figure S4B). This data indicated that active actin remodeling may occur following *LINC01133* knockdown. Therefore, we further evaluated the expression and/or activity of some proteins involved in the regulation of actin filaments, stress fibers formation and focal adhesions such as TESK1 and Cofilin. TESK1 phosphorylates and thereby inactivates the Actin severing protein Cofilin at Ser3, thus regulating the organization of the Actin cytoskeleton [29]. We identified *TESK1* as being differentially expressed in 12Z knockdown cells by RNA-seq (Table S4) and confirmed that the protein was significantly increased following *LINC01133* knockdown (1.5-times higher, adjp < 0.05) (Figure 5B). These changes in TESK1 expression were associated with a significant increase in Cofilin phosphorylation to 2.2-fold higher (adjp < 0.05) following *LINC01133* knockdown (Figure 5C). Together this data indicates that *LINC01133* may regulate actin remodeling in endometriosis epithelial cells via this pathway.

**Figure 5.** *LINC01133* knockdown affects cellular morphology of 12Z cells (**A**) Immunofluorescence of F-actin 72 h after transfection with siRNA control and 2 different *LINC01133* oligos shows a more flattened phenotype and an increase in the number of actin stress fibers. Representative confocal images are shown (three independent experiments were analyzed). Cell nuclei were visualized with DAPI. The magnification scale of 50 µm is indicated with white line on the figure. (**B**) *LINC01133* knockdown leads to upregulation of the levels of TESK1 protein. Left: Representative Western blot showing TESK1 levels together with the α-tubulin loading control. Right: TESK1 relative protein levels normalized to α-tubulin determined by densitometric analysis of Western blots. The relative levels of TESK1 from three independent experiments are shown as bar graphs with the control set to 1 and the mean and + SD of protein shown (**C**) *LINC01133* knockdown increases the levels of Cofilin phosphorylation. Left: Representative example of *p*-Cofilin and Cofilin levels detected by Western blot with α-tubulin loading control. Right: Phosphorylated Cofilin relative levels normalized to α-tubulin determined by densitometric analysis of Western blots. The relative levels of phosphorylated Cofilin from three independent experiments are shown as bar graphs with the control set to 1, and the mean and +SD of protein shown. Statistically significant differences between the groups are indicated by adj *p*-values on the top of each graph (ANOVA, with Dunnett's multiple comparison test). **Figure 5.** *LINC01133* knockdown affects cellular morphology of 12Z cells (**A**) Immunofluorescence of F-actin 72 h after transfection with siRNA control and 2 different *LINC01133* oligos shows a more flattened phenotype and an increase in the number of actin stress fibers. Representative confocal images are shown (three independent experiments were analyzed). Cell nuclei were visualized with DAPI. The magnification scale of 50 µm is indicated with white line on the figure. (**B**) *LINC01133* knockdown leads to upregulation of the levels of TESK1 protein. Left: Representative Western blot showing TESK1 levels together with the α-tubulin loading control. Right: TESK1 relative protein levels normalized to α-tubulin determined by densitometric analysis of Western blots. The relative levels of TESK1 from three independent experiments are shown as bar graphs with the control set to 1 and the mean and + SD of protein shown (**C**) *LINC01133* knockdown increases the levels of Cofilin phosphorylation. Left: Representative example of *p*-Cofilin and Cofilin levels detected by Western blot with α-tubulin loading control. Right: Phosphorylated Cofilin relative levels normalized to α-tubulin determined by densitometric analysis of Western blots. The relative levels of phosphorylated Cofilin from three independent experiments are shown as bar graphs with the control set to 1, and the mean and +SD of protein shown. Statistically significant differences between the groups are indicated by adj *p*-values on the top of each graph (ANOVA, with Dunnett's multiple comparison test).

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