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. 2016 Aug 29;139(11):2570–2582. doi: 10.1002/ijc.30386

Figure 2.

Figure 2

(a) siRNA mediated knockdown of FAT1 expression in glioma cell lines under hypoxia: siRNA (siControl and siFAT1) transfection was done in U87MG, U373MG and GOS3 cell lines maintained under hypoxia. FAT1 mRNA level was checked after 72 hr of siRNA transfection. We found >80% downregulation of FAT1 mRNA in siFAT1 transfected cells as compared to siControl transfected cells under hypoxic condition, 72 hr post‐transfection. 18S was used as internal control. All the experiments were done in triplicates and repeated thrice. (b,c) Effect of FAT1 knockdown on HIF1α expression in glioma cell lines: HIF1α mRNA and protein level were assessed, 72 hr post‐siFAT1 transfection, under hypoxic condition by q‐PCR and Western blot respectively. (b) At mRNA level, on FAT1 knockdown, HIF1α showed 65% and 37% reduction in U87MG and U373MG cells respectively under hypoxic condition. (c) Similarly, at protein level, significant decrease in HIF1α level in both siFAT1 treated U87MG and U373MG cells as compared to siControl treated cells was found. However, in GOS3, there was no significant difference in HIF1α at both mRNA and protein level between cells treated with control siRNA and those treated with FAT1 siRNA (b,c). 18S was used as internal control for q‐PCR and β‐actin was used as loading control for Western blot. Nor: Normoxia, Hyp siCont: Hypoxia siControl, Hyp siFAT1: Hypoxia siFAT1 (d) FAT1 expression after HIF1α knockdown in U87MG glioma cell line: FAT1 mRNA expression was checked by q‐PCR in U87MG cells 72 hr post transfection of HIF1α siRNA under hypoxic condition. On HIF1α knockdown, FAT1 expression was observed as comparable to Control siRNA treated cells. 18S was used as internal control. (e) Effect of FAT1 knockdown on HIF1α activity in U87MG glioma cell line maintained under hypoxia: HIF1α promoter luciferase assay was done to assess HIF1α transcriptional activity after FAT1 knockdown under hypoxic condition. After 24 hr of siFAT1 transfection, cells were transfected with 1 µg of HIF1α reporter plasmid. Luciferase activity was assayed 48 hr post‐HIF1α reporter plasmid transfection. The luciferase activity observed in cells transfected with pGL3 vector was taken as control and set to 1 and the activity observed in the cells co‐transfected with control siRNA/FAT1 siRNA and wildtype promoter construct was expressed as fold change. On FAT1 knockdown, 60% decrease (p = 0.01) in HIF1α promoter luciferase activity was observed in comparison to siControl treated cells, as determined by Student's t test. The experiment was repeated thrice and results are expressed as mean ± standard deviation. As mentioned earlier, the first two lanes for GOS3 in Figure 2c are also depicted in Figure 1d.