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Fig. 6 | Molecular Cancer

Fig. 6

From: A novel protein encoded by circINSIG1 reprograms cholesterol metabolism by promoting the ubiquitin-dependent degradation of INSIG1 in colorectal cancer

Fig. 6

Hypoxia-induced EIF4A3 promotes circINSIG1 expression. A The EIF4A3 binding sites on the upstream and downstream of the back-splicing site of circINSIG1 predicted by the CircInteractome. B Western blot analysis of EIF4A3 expression in HCT8 and DLD1 cells under hypoxia. C The correlation between EIF4A3 and circINSIG1 expression in HCT8 and DLD1 under hypoxia. D Western blot analysis of RNA pull-down assays with EIF4A3 antibody. E RIP-qPCR assay in HCT8 cells transfected with or without wild-type (WT) circINSIG1 minigene using the indicated primers. IgG served as a control. F Schematic of circINSIG1 minigenes with WT or mutant (mut) EIF4A3 binding sites on the flanked intron regions of INSIG1. G HCT8 cells with or without EIF4A3 overexpression were transfected with WT or various mutant circINSIG1 minigenes. qRT-PCR analyzed the expression level of circINSIG1 in HCT8 cells. H qRT-PCR analysis of circINSIG1 expression in HCT8 and DLD1 cells with EIF4A3 overexpression. I qRT-PCR analysis of circINSIG1 expression in HCT8 and DLD1 cells with EIF4A3 knockdown. J-l HCT8 cells with or without EIF4A3 overexpression were transfected with shRNA targeting circINSIG1 (shcircINSIG1) for rescue assays. Free cholesterol content detection was detected. (J) Western blot analysis of indicated protein expression. (K) qRT-PCR analysis of SREBP2 target genes. (L) Values are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA

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