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Fig. 3 | Molecular Cancer

Fig. 3

From: CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis

Fig. 3

CircPPAP2B directly interacts with HNRNPC in a m6A-dependent manner A. RNA pulldown assay was performed using a biotin-labeled sense or antisense probe of circPPAP2B. The proteins enriched by sense or antisense probe of circPPAP2B were separated by SDS-PAGE and subjected to silver staining. B Mass spectrometry analysis identified HNRNPC as one of the most abundant proteins enriched by the sense probe of circPPAP2B. C RNA pulldown assay was performed to confirm the direct interaction between circPPAP2B and HNRNPC in ccRCC cell lines. D RNA pulldown assay was performed to confirm the direct interaction between circPPAP2B and HNRNPC in 293 T. E RIP assay was performed to validate direct interaction between circPPAP2B and HNRNPC in ccRCC cell lines using anti-HNRNPC antibody. F RNA FISH was performed to determine the co-localization of circPPAP2B and HNRNPC in ccRCC cell lines. G Full-length and truncated flag-labeled HNRNPC was constructed and RIP assays were performed to identify the specific domain of HNRNPC which interacts with circPPAP2B. H RNA pulldown assays were performed to identify the specific domain of HNRNPC which interacts with circPPAP2B. I MeRIP assays were performed to investigate whether circPPAP2B undergoes m6A modification. J Graphic illustration of the secondary structure of circPPAP2B and its m6A modification site predicted by RNA-fold software. Construction of WT and m6A modification site mutant circPPAP2B. K RNA pulldown assay and dot blot assay were performed to validate the m6A modification site in circPPAP2B. L RIP assay was performed to validate the m6A modification site in circPPAP2B and its role in the interaction between circPPAP2B and HNRNPC. M Transwell assay was performed on the role of m6A modification site in circPPAP2B in promoting ccRCC invasion. I The representative images of lung metastases in nude mice by HE staining. Tumor burdens were reflected by the percentages of tumor area in each slide. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT group

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Molecular Cancer

ISSN: 1476-4598