Fig. 5

CircPPAP2B modulates the interaction between HNRNPC and splicing factors to regulate pre-mRNA alternative splicing A GSEA enrichment analysis was performed to reveal the relationship between HNRNPC and RNA splicing with mRNA processing in ccRCC. B-C Mass Spectrometry analysis and STRING prediction were employed to identify splicing factors HNRNPC regulated in ccRCC. D CoIP assay was performed to confirm the direct interaction between PTBP1, HNRNPK, HNRNPL, HNRNPA2B1, and HNRNPC. E CoIP assay was performed to confirm whether circPPAP2B overexpression influenced the interaction between HNRNPC and PTBP1 or HNRNPK. F CoIP assay was performed to confirm whether circPPAP2B knockdown influenced the interaction between HNRNPC and PTBP1 or HNRNPK. G Alternative splicing events were identified in both circPPAP2B knockdown and anti-HNRNPC circPPAP2B knockdown ccRCC cells, including skipped exon (SE), alternative 5’ splice site (A5SS), alternative 3’ splice site (A3SS), mutually exclusive exons (MXE) and retained intron. H 235 alternative splicing genes were shared in both circPPAP2B knockdown and anti-HNRNPC circPPAP2B knockdown ccRCC cells. I GO enrichment analysis identified enriched pathways with shared alternative splicing genes. J The Pie diagram shows the ratio of different types of alternative splicing events regulated by circPPAP2B knockdown. K The Venn diagram showed 40 alternative splicing events were shared in both circPPAP2B knockdown and anti-HNRNPC circPPAP2B knockdown ccRCC cells. L Alternative splicing events of CD44 were identified in the anti-HNRNPC RIP-sequencing assay. M qPCR assay was performed to validate the selected alternative of CD44 in OE-NC and OE-circPPAP2B ccRCC cells. N RIP-qPCR with anti-HNRNPK antibody was performed to validate the interaction between HNRNPK and CD44 alternative splicing