Phosphoproteomics analysis of PIK3R1 KO cells reveals signaling responses to Notch inhibition. (A) Significantly enriched KEGG pathways of proteins with altered phosphorylation sites, comparing PIK3R1 KO CB-103–treated vs NT control CB-103–treated RPMI-8402 cells. Top 30 pathways are shown, solid line color scale indicates adjusted P value, dot size of leading edge displays percentage of genes enriched in corresponding pathways. (B) Kinase substrate–enrichment analysis (KSEA) of phosphorylation profiles, comparing PIK3R1 KO CB-103–treated vs NT CB-103–treated RPMI-8402 cells. Red, kinases with positive z score; blue, kinases with negative z score. (C) Interactions among phosphoproteins within 4 of the top enriched KEGG pathways in panel A: assessing cell cycle, AMPK signaling, cellular senescence, and EGFR tyrosine kinase inhibitor resistance pathways. Line color indicates the strength of interaction (“Confidence” from the STRING database). Key nodes are gated with black rectangle. (D) Detailed plots of key phosphoproteins with annotated phosphosites and corresponding FCs from rectangle area in panel C. Red circle indicates phosphoproteins with phosphorylation changes as log₂FC > 1; false discovery rate < 0.05. Gray circle indicates phosphoproteins with phosphorylation sites omitted. Red connecting line, protein interaction from STRING database. Gray radiating line, detailed phosphorylation sites associated with phosphoproteins. (E) Total protein and phosphorylation level of indicated phosphosites by western blotting for key proteins involved in the indicated nodes or pathways: Notch signaling (light blue); AKT (red); S6K and S6 (green); RB (orange); prosurvival signaling (dark blue) in RPMI-8402 NT and PIK3R1 KO cells in response to CB-103. TBP was used as loading control. (F) Model summarizing the key nodes of Notch signaling–inhibition resistance mechanism caused by the loss of PIK3R1. TBP, TATA box-binding protein.