Fig 1: Overexpression of circFNDC3B or RNF41 suppresses tumor growth, stemness and liver metastasis via modulating ASB6 in vivo.A Photographs of xenograft tumors. B Quantitative analysis of tumor volume. C Quantitative analysis of tumor weight. D–H The mRNA levels of RNF41, OCT4, Nanog, SOX2, and CD133 in xenograft tumors were detected by qRT-PCR. I The protein levels of ASB6, OCT4, Nanog, SOX2, and CD133 were detected by western blotting with quantitative analysis. J The immunoreactivities of Ki-67, CD133, and ASB6 in xenograft tumors were detected by IHC analysis. K Representative photographs of liver tissues derived from in vivo liver metastasis mice model. L The histological changes of liver tissues were detected by H&E staining. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: circFNDC3B increases RNF41 mRNA stability and expression via binding to FXR2.A The RNF41 level in CRC tissues was detected by qRT-PCR. B The correlation between circFNDC3B and RNF41 level in CRC tissues was analyzed by spearman correlation analysis. C The interaction between biotinylated circFNDC3B and FXR2 was detected by RNA pull-down assay. Random probe acted as a negative control. D The interaction between circFNDC3B and FXR2 was detected by RIP assay. Normal IgG served as a negative control. E The co-localization of circFNDC3B and FXR2 was detected by RNA FISH and immunofluorescence (IF). Scale bar, 10 μm; Green, circFNDC3B; Red, FXR2; Blue, DAPI. F, G The interaction between RNF41 and FXR2 was detected by RIP assay. Normal IgG served as a negative control. H The mRNA stability of RNF41 was determined by RNA stability assay. I, J The mRNA and protein levels of RNF41 in transfected CRC cells were detected by qRT-PCR and Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Knockdown of RNF41 counteracts circFNDC3B-suppressed CRC stemness and metastasis.A–E The expression of stemness markers were detected by qRT-PCR and Western blotting. F Representative sphere images of transfected CRC cells with quantitative analysis. G CD133+ cells were analyzed by flow cytometry with quantitative analysis in transfected CRC cells. H Cell migration was monitored by wound healing assay with quantitative analysis in transfected CRC cells. I Cell invasion was measured by Transwell invasion assay with quantitative analysis. *P < 0.05, **P < 0.01.
Fig 4: Overexpression of ASB6 reverses circFNDC3B or RNF41-mediated regulation of CRC stemness and metastasis.A–E The expression of stemness markers were detected by qRT-PCR and western blotting. F Representative sphere images of transfected CRC cells with quantitative analysis. G CD133+ cells were analyzed by flow cytometry with quantitative analysis in transfected CRC cells. H Cell migration was monitored by wound healing assay with quantitative analysis in transfected CRC cells. I Cell invasion was measured by Transwell invasion assay with quantitative analysis. *P < 0.05, **P < 0.01.
Fig 5: circFNDC3B promotes ASB6 degradation through RNF41-mediated ubiquitination.A The protein level of ASB6 was determined by western blotting with quantitative analysis. B The ubiquitination of ASB6 was assessed by co-IP in transfected CRC cells. C The interaction between ASB6 and RNF41 was detected by Co-IP. Normal IgG served as a negative control. D The protein levels of RNF41 and ASB6 were determined by western blotting with quantitative analysis in transfected CRC cells. E The protein level of ASB6 was determined by western blotting with quantitative analysis in transfected CRC cells. F The interaction between ASB6 and RNF41 in circFNDC3B-overexpressing cells was detected by Co-IP. *P < 0.05, **P < 0.01.
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