Fig. 6
PDE5A-500aa interaction with PIK3IP1 inhibits the PI3K/AKT pathway. A Volcano plots comparing fold changes in gene expression in vector and circPDE5A overexpressing KYSE30 cells. B Bubble plots of KEGG pathway enrichment analysis showing the top 20 pathways significantly altered in circPDE5A overexpressing KYSE30 cells. C Circle plots showing the top 10 significantly altered pathways that were up- or down-regulated in circPDE5A overexpressing KYSE30 cells. D Immunoprecipitation (IP) of IgG or anti-flag antibodies in 293 T cells overexpressing circPDE5A-flag, and pull-down proteins visualized by SDS-PAGE and silver staining. E Identification of PDE5A-500aa binding proteins by LC–MS/MS analysis. Upper panel: Proteins pulled down by IP with IgG or anti-flag antibodies. LC–MS/MS analysis is used to identify potential proteins specifically binding to PDE5A-500aa. Bottom panel: LC–MS/MS identifies PIK3IP1 protein, a PI3K/AKT pathway regulator, as part of the PDE5A-500aa protein complex. F Co-IP shows the interaction between PDE5A-500aa and PIK3IP1. G Visualization of docking patterns and key residue interfaces of PDE5A-500aa with PIK3IP1 obtained by PyMOL software. H Co-localization of PDE5A-500aa and PIK3IP1 proteins in the cytoplasm of ESCC cells examined by immunofluorescence. Bar represents 5 μm. I Levels of PIK3IP1 and PI3K/AKT downstream target proteins in KYSE30 cells detected by WB after treatment with vector, circPDE5A-Mut, circPDE5A, and circPDE5A + si-PIK3IP1 plasmid, respectively. J Levels of PIK3IP1 and PI3K/AKT downstream target proteins in ECA109 cells detected by WB after treatment with sh-NC, sh-circPDE5A-1, sh-circPDE5A-2, and sh-circPDE5A + OE-PIK3IP1 plasmid, respectively. K–L Immunohistochemical (IHC) detection of PIK3IP1 protein level in ESCC and adjacent normal tissues. Bar represents 20 μm. M Correlation analysis between PDE5A-500aa and PIK3IP1 protein level in ESCC tissues. N–P IHC detection of PIK3IP1 protein levels in ESCC tissues with different T (N), N (O), and TNM (P) stages. Q Kaplan–Meier analysis of the correlation between PIK3IP1 protein level and OS in patients with ESCC. ***P < 0.005