Fig. 5.

Effect of NFкB pathway activation on FAT1 promoter activity and endogenous FAT1 expression. NFкB pathway activation was done by treating GBM cells with known NFкB activators (severe hypoxia, TNFα and NFкB expression vector). a FAT1 promoter activity under severe hypoxia. GBM cells transfected with pGL3F1 were exposed to severe hypoxia (0.2% O₂) and normoxia (20% O₂) for 48 h. Luciferase activity of pGL3F1 was significantly increased under severe hypoxia as compared to normoxic control U87MG. b FAT1 mRNA expression under severe hypoxia. Glioma cells exposed to severe hypoxia showed significant increase in FAT1 mRNA expression along with increased NFкB (RelA) expression in both U87MG and A172 as compared to the respective normoxic cells. c FAT1 promoter activity in TNFα treated glioma cells. GBM cells were transfected with pGL3F1 and treated with TNFα (100 ng/ml) for 24 h followed by FAT1 promoter activity and mRNA expression analyses. Significant increase in pGL3F1 luciferase activity was observed in U87MG cells treated with TNFα as compared to the control (untreated cells). d FAT1 mRNA expression in TNFα treated glioma cells. Glioma cells were treated with TNFα as described above. Increased endogenous FAT1 mRNA expression in both U87MG and A172 was seen along with increased NFкB (RelA) expression, compared to the untreated control cells. e FAT1 promoter activity in glioma cells with overexpressed NFкB (RelA). GBM cells were co-transfected with pGL3F1 + RelA+IKBK and control vector (pGL3B + eBABE) for 48 h followed by FAT1 promoter activity and mRNA expression analyses. Significantly increased FAT1 promoter activity was observed in the cells co-transfected with RelA+ IKBK expression vector as compared to the control cells. f FAT1 mRNA expression in glioma cells with overexpressed NFкB (RelA). U87MG and A172 cells co-transfected with RelA+ IKBK showed increased mRNA expression of FAT1 and NFкB (RelA) expression as compared to the control cells. Bar graph represents fold luciferase activity of FAT1 promoter constructs (normalized to pGL3Basic and internal control pRLTK) and fold mRNA expression. All experiments were done in triplicates and repeated thrice. Statistical analysis was performed using a paired two-tailed Student’s t-test. P-value < 0.05 was taken as significant (*p < 0.05, **p < 0.01, ***p < 0.001)