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Fig. 4 | Molecular Cancer

Fig. 4

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

Fig. 4

circINSIG1-121 interacts with INSIG1 to promote ubiquitin-dependent degradation of INSIG1 to induce cholesterol biosynthesis. A qRT-PCR analysis of INSIG1 expression in HCT8 cells with circINSIG1 or circINSIG1-121 overexpression. B Western blot analysis of INSIG1 expression in HCT8 and DLD1 cells with circINSIG1 or circINSIG1-121 overexpression. C Immunofluorescence assay showed the colocalization of circINSIG1-121 and INSIG1 in HCT8 cells. Scale bar = 10 μm. D Immunoprecipitation assays using circINSIG1-121 or INSIG1 antibodies confirmed the interaction between circINSIG1-121 and INSIG1 in HCT8 cells. E Western blot analysis of INSIG1 expression in HCT8 transfected with circINSIG1 and treated with MG132 (10 µM) or 3-Methyladenine (3-MA) (10 mg/ml). F Western blot analysis of INSIG1 ubiquitin levels after immunoprecipitation using INSIG1 antibody in HCT8 cells transfected with circINSIG1 or circINSIG1-121 vector. G Western blot analysis of full-length SREBPs (fSREBPs) and active nuclear SREBPs (nSREBPs) protein levels in HCT8 cells transfected with the indicated plasmids. H qRT-PCR analysis of SREBP2 target genes in HCT8 cells with circINSIG1 or circINSIG1-121 overexpression. I Free cholesterol content detection in HCT8 cells with circINSIG1 or circINSIG1-121 overexpression. Values are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by one-way ANOVA

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Molecular Cancer

ISSN: 1476-4598