Journal: bioRxiv

Article Title: Proteomic Analysis of PTEN-Deficient Cells Reveals Src-Mediated Upregulation of EphA2 and Therapeutic Potential of Dual Inhibition

doi: 10.1101/2025.03.13.643053

Figure Lengend Snippet: (A) Differentially regulated Src interacting phosphoproteins were identified using BioGRID database (version 4.4), and the protein-protein interaction map was plotted using Cytoscape. Phosphorylated protein interactors marked in red were significantly upregulated in PTEN KO cells, while phosphorylated protein interactors marked in blue were significantly downregulated. The darker the color, the bigger the difference. The node size corresponded to p value, and bigger text size indicated smaller p value. One representative phosphorylation site of each protein was labeled for the corresponding proteins. The full list of differentially regulated phosphosites is shown in Supplemental Data 7 . The edges with arrows indicate that these proteins are the substrates of Src. (B-C) PTEN knockout ( B ) and transient knockdown ( C ) in MCF10A cells induced Src-Y416 hyperphosphorylation. (D-E) EphA2 expression levels were measured in PTEN deficient MCF10A-PTEN-KD cells ( D ), HCC1937 and SPAC-1-L cells ( E ) with siRNA knockdown of Src. ( F-H ) Src inhibitor Dasatinib was used to treat MCF10A cells with PTEN knockout (F) , MCF10A cells ( G ) and PEO1 cells with PTEN transient knockdown ( H ). Src pY416 was used to evaluate the dasatinib inhibition efficiency. ( I ) EphA2 expression was checked by western blot in PTEN deficient HCC1937, SNGM and SPAC-1-L cells treated with dasatinib. (J-L) EphA2 expression was reduced by MEK inhibitors U0126 and Trametinib in MCF10A cells with PTEN knockout (J) , in MCF10A cells ( K ) and MCF7 cells ( L ) with transient siPTEN knockdown. Antibody targeting phosphorylated ERK1/2 at T202/Y204 was employed to evaluate the activation of MEK and the successful inhibition of MEK by kinase inhibitors U0126 and Trametinib.

Article Snippet: Anti-AKT pS473 (4060T), anti-total-AKT (4691S), anti-ERK1/2 pT202/Y204 (4370S), anti-total ERK1/2 (4695S), anti-EphA2 (6997S), anti-SPHK1 (12071), anti-DKK1 (48367), anti-TGFBR2 (41896), anti-Rab27B (44813), anti-Src_pY416 (6943S), anti-total-Src (2109S), anti-P70S6K_pT389 (97596S), anti-GSK-3α/β pSer21/9(9331S), anti-PARP(9542T), Caspase 3 (14220T), Cleaved Caspase 3 (9664T), and anti-actin (4970L) antibodies were purchased from Cell Signaling Technology (CST).

Techniques: Labeling, Knock-Out, Knockdown, Expressing, Inhibition, Western Blot, Activation Assay