Fig 2: a. Relative expression of FAT4 in MCAS cells following its knockdown at a. mRNA level, and b. protein levels. FAT4 was knocked down significantly (p = 0.0016) in MCAS. Full-length blots are presented in Supplementary figure S1. c. Relative expression of FAT4 in OVSAHO cells following its knockdown at a. mRNA level, and d. protein levels. Full-length blots are presented in Supplementary figure S1. FAT4 was knocked down significantly in OVSAHO (p = 0.0055). Data represent mean and standard deviation from at least three independent experiments performed in triplicates

Fig 3: a. Relative expression of FAT4 at mRNA levels in ovarian cancer cell lines. MCAS and OVSAHO show high FAT4 expression as compared to A2780 and A2780 cis (p = 0.0001). b. Relative expression of FAT4 at protein levels in MCAS and OVSAHO cells. FAT4 was highly expressed in OVSAHO (p = 0.0459), and MCAS (p = 0.0107), cells while, it was least expressed in A2780 cis (p = 0.006) and A2780 (p = 0.0012) cells. Full-length blots are presented in Supplementary figure S1. c. Differential expression of FAT4 in tumor and healthy ovarian tissue sample from GEPIA database. FAT4 was significantly downregulated in 426 tumor samples as compared to 87 healthy samples. Red line represents tumor samples and green line represents healthy ovarian samples. Data represent mean and standard deviation from at least three independent experiments performed in triplicates

Fig 4: a. FAT4 knockdown upregulated the expression of E2F5 suggesting a link between the two genes. b. A suggested mechanism of EMT and cell cycle regulation by FAT4 based on Western blot obtained data. Inhibition of FAT4 regulates the expression of EMT markers and promotes cell cycle progression via the suggested pathway. The solid lines indicate the results obtained from the previous study [13], while the dotted lines indicate results of our western blotting experiments

Fig 5: Regulatory effects of FAT4 on the expression of proteins involved in EMT, Hippo, Wnt-β-catenin, apoptotic, and retinoblastoma pathways by Western blotting. a. Relative expression variation of proteins. The expression of Vimentin (p = 0.0001), YAP (p = 0.0018), β-catenin (p = 0.001), Bcl2 (p = 0.0001), cyclin D1 (p = 0.0017) and cdk4 (p = 0.0025) was higher in FAT4 siRNA treated cells as compared to control. β-actin was used as an internal control. b. Western blot demonstrating bands for protein expression in control and FAT4 knocked cells. Full-length blots are presented in Supplementary figure S1. c. The ratio of phosphorylated to total YAP, GSK-3β, β-catenin, and retinoblastoma proteins following FAT4 repression. The pYAP/ YAP ratio was lower in FAT4 siRNA treated cells as compared to the control (p = 0.0286). Similarly, pGSK-3-β/GSK-3-β ratio, and pβ-catenin/β-catenin ratio was lower in FAT4 siRNA treated cells (p = 0.018, and p = 0.001 respectively) as compared to control. There was no significant difference in pRb/Rb ratio between FAT4 siRNA treated cells and control. MCAS cells treated with scrambled siRNA was used as control. Data represent mean and standard deviation from at least three independent experiments performed in triplicates