Fig. 6

COMP induces EMT and cross-talks with β-catenin signaling pathway. The mRNA expression levels of EMT-specific genes in A SKOV3 and B OAW42 cells treated with COMP (20 μg/ml) or BSA (20 μg/ml) were analyzed using a PrimePCR Custom Plate. Volcano plots with a Log2 scale on the X-axis and a Log10 scale on the Y-axis were used to represent the data. GAPDH was used as a reference gene, and the relative mRNA expression was calculated using the 2^−ΔΔCt method. Multiple unpaired t-tests were used for statistical analyses. C, D RT-qPCR analysis of EMT marker genes, in SKOV3 and OAW42 cells treated with COMP (20 and 50 μg/ml) or BSA (50 μg/ml) as a control. GAPDH was used as a reference gene, and the relative mRNA expression was calculated using the 2^−ΔΔCt method. The p-value was calculated by two-way ANOVA followed by Tukey’s post-test. E, F RT-qPCR analysis of CDH2 and FN1 expression in SKOV3 cells treated with DAPT (1 μM), COMP (50 μg/ml), and DAPT + COMP 20 μg/ml and 50 μg/ml. GAPDH was used as a reference gene and the relative mRNA expression was calculated using the 2^−ΔΔCt method. The p-values were calculated by one-way ANOVA followed by Dunnett’s post-test. G RT-qPCR analysis of CTTNB1 (β-catenin) in SKOV3 cells in the presence of COMP (20 and 50 μg/ml) or BSA (50 μg/ml) as a control. GAPDH was used as a reference gene, and the relative mRNA expression was calculated using the 2^−ΔΔCt method. The p-value was calculated by one-way ANOVA followed by Tukey’s post-test. H Analysis of protein expression levels of active (non-phosphorylated) β-catenin in SKOV3 cells treated with COMP (20 and 50 μg/ml) or BSA (50 μg/ml) as a control by western blotting. The p-value was calculated by one-way ANOVA followed by Dunnett’s post-test. I Dual-Luciferase reporter assay measuring the activity of β-catenin in SKOV3 cells treated with BSA (50 μg/ml), BSA + DAPT (1 μM), COMP (50 μg/ml), and COMP + DAPT (1 μM). Untreated cells were used as a control. The p-value was calculated by one-way ANOVA followed by Dunnett’s post-test. J Graphical representation of the proposed molecular mechanism of COMP paracrine action. Stromal expression of COMP likely by CAFs in ovarian TME bridges Notch3-jagged1 interaction, leading to EMT induction and β-catenin activation. The data are representative of at least three independent experiments and graphs depict the mean with the standard deviation. The significance threshold was set at p < 0.05. (*, **, ***, ****, and ns indicate p < 0.05, p < 0.01, p < 0.001, p < 0.0001, and non-significant, respectively)