Fig 2: Transcriptome-wide RNA-Seq and m6A-Seq assays identify potential targets of VHL involved m6A modification.(A) Schematic flow chart demonstrating the framework to identify VHL-involved m6A targets. (B) Overlapping analysis of genes identified by different m6A peaks (DM) and differentially expressed genes (DEGs). (C) Overlapping analysis of genes identified by Ctrl only m6A site genes and DEGs. (D) Overlapping analysis of top enriched genes by strategy 1 and strategy 2. (E) Heatmap of DEGs and different m6A peaks (DM) of top enriched genes. (F) Clustergram of top enriched genes in KEGG pathways. (G) Genome browser representative tracks of 3 biological replicates displaying the m6A read distribution and changes (based on the m6A MeRIP-Seq and RNA-Seq data) in PIK3R3 transcripts upon the KO of VHL in HKC cells. (H) qPCR analysis of PIK3R3 mRNA levels in HKC Ctrlsg and VHLsg cells. (I) MeRIP-qPCR analysis of PIK3R3 m6A levels in HKC Ctrlsg and VHLsg cells. (J) The association of METTL3 with PIK3R3 mRNA was assessed by METTL3 RIP-qPCR analysis. Data show mean ± SD, ***P < 0.001, ****P < 0.0001, 1-way ANOVA analysis.

Fig 3: PIK3R3 suppresses AKT signaling.(A–D) Immunoblotting analysis shows increased AKT phosphorylation in PIK3R3-KO HKC cells (A) or decreased AKT phosphorylation in PIK3R3 overexpressing cell lines (B–D). pAKT(T308), pAKT(S473), AKT, and PIK3R3 blots were run in parallel using the same biological samples. (E) Immunoblotting with lysate of tumors grown out from mice renal subcapsule orthotopic injected empty vector or PIK3R3-V5 transduced UMRC2 luciferase cell lines. pAKT(T308), pAKT(S473), AKT, and PIK3R3 blots were run in parallel using the same biological samples. (F–I) Immunoblotting analysis shows increased p85 in PIK3R3-KO HKC cells (F) or decreased p85 in PIK3R3 overexpressing cell lines (G–I). P110-α and P110-β blots were run in parallel using the same biological samples.

Fig 4: PIK3R3 suppresses tumor growth in vivo.(A–D) Representative bioluminescence imaging (A) of mice renal subcapsule orthotopic injected with Ctrlsg or PIK3R3sg transduced HKC luciferase cell lines and corresponding quantitation data of bioluminescence signal (B), tumor weight (C), or tumors images (D) after dissection. (E–H) Representative bioluminescence imaging (E) of mice renal subcapsule orthotopic injected with empty vector or PIK3R3-V5 transduced UMRC2 luciferase cell lines and corresponding quantitation data of bioluminescence signal (F), tumor weight (G), tumors images (H) after dissection. Data show SEM for B and F or mean ± SD for others; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA (B and F), 1-way ANOVA (C) or unpaired t test (G).

Fig 5: PIK3R3 is overexpressed and p21 is underexpressed in colorectal cancer.a Expression analysis of PIK3R3 in CRC tissues and normal intestinal tissues from a dataset (GSE21510) of GEO database. b Gene ontology (GO) analysis of PIK3R3 co-expression genes. c Correlation analysis of PIK3R3 and p21 in CRC tissues from the dataset (GSE21510). d IHC staining of typical samples from CRC and adjacent tissues. e Quantification of PIK3R3 and p21 expression in tumor groups and normal epithelial groups. f The protein levels of PIK3R3 and p21 in CRC and adjacent normal tissues. These numeral results were displayed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, t-test.