Figure 5

Decreased SUSD2 levels increased mRNA associated with EMT. (a) RT-qPCR analysis of EMT mRNA markers in HGSOC cells (OVCAR3, OVSAHO and KURAMOCHI cell lines). RNA was isolated from HGSOC cell line monolayers and OVCAR3 spheroids, and the generated cDNA was used as a template for RT-qPCR. The graphs depict two separate experiments (indicated by the separation of genes in the y axis) investigating the effect of SUSD2 levels on mRNA expression of several well-characterized EMT genes (N-cadherin, ZEB1, Twist1, E-Cadherin KRT14,STEAP1, AHNAK, SNAI1, COL5A2 and SNAI3). Fold changes were calculated by comparing the levels of mRNA expression observed in KD cell lines to NT cell lines (Ct values were calculated via the comparative Ct method). Each experiment was performed in triplicate. P-values were derived using a standard two-tailed t-test between SUSD2-KD cells and NT controls. (b) Morphology of KURAMOCHI cells. Macroscopic images of adherent KURAMOCHI cells were taken using bright field light microscopy via a high-powered objective (× 400 magnification). KURAMOCHI NT cells are pictured on the left (cells expressing endogenous levels of SUSD2 protein) and KURAMOCHI sh4-4 cells are pictured on the right (cells stably transfected with functional SUSD2-targeting shRNA). Black arrows indicate elongations from filopodia formation evident in the KURAMOCHI sh4-4 cells.