Fig 2: CEP inhibits the PI3K/Akt/mTOR pathway and promotes the ubiquitin-proteasomal degradation of p70S6K. A-B Western blot analysis of total and phosphorylated PI3K, AKT1, mTOR, p70S6K, and S6 in KYSE150, KYSE150R (A), TE-1, and KYSE410 (B) cells. Cells were treated with vehicle or CEP, in the presence or absence of irradiation (IR). β-actin served as the loading control. C Cycloheximide (CHX) chase assay evaluating p70S6K protein stability. Cells were treated with CHX in the presence or absence of CEP for the indicated times (0, 2, 4, 8, and 12 h) prior to immunoblotting. D Western blot analysis evaluating the rescue effect of proteasome inhibition. Cells were pre-treated with the proteasome inhibitor MG132 followed by CEP treatment. E Cell-based ubiquitination assay demonstrating CEP-induced polyubiquitination of p70S6K. Cells co-transfected with p70S6K and HA-tagged ubiquitin (HA-Ub) plasmids were treated with CEP or vehicle in the presence of MG132. Cell lysates were immunoprecipitated (IP) with an anti-p70S6K antibody and immunoblotted (IB) with an anti-HA antibody. Input lysates were analyzed for baseline protein expression

Fig 3: p70S6K mediates CEP-induced metabolic suppression, depending on the Q347 binding pocket. A Western blot analysis of p70S6K, Phospho-p70S6K, and downstream apoptosis-related proteins in OE-NC and OE-p70S6K cells following CEP exposure. β-actin served as the loading control. B Cell viability of KYSE150 and KYSE150R cells transfected with empty vector (OE-NC) or wild-type p70S6K (OE-p70S6K), followed by treatment with vehicle or CEP. C-D Representative images (C) and quantification (D) of colony formation assays in the indicated cell groups. E-F Apoptosis rates quantified by bar charts (E) and representative flow cytometry plots (F) of cells treated with or without CEP under p70S6K overexpression. G Real-time metabolic flux analysis measuring the ECAR and OCR in OE-NC and OE-p70S6K cells. H CETSA evaluating the thermal stability of WT and Q347A mutant p70S6K proteins in the presence of vehicle or CEP across a temperature gradient. I Immunoblot analysis of downstream S6 phosphorylation (p-S6) and total S6 in KYSE150R cells transfected with empty vector, wild-type p70S6K (WT-OE), or Q347A mutant p70S6K (Mut-OE) plasmids, followed by DMSO or CEP treatment. Flag-tag confirms the expression of the exogenous p70S6K constructs, and β-actin serves as the loading control. J-K Representative images (J) and relative quantification (K) of clonogenic survival. OE-WT and OE-Q347A cells were treated with vehicle or CEP prior to a single fixed dose of irradiation (4 Gy). Data are normalized to the OE-WT+4 Gy group. L ECAR and OCR profiles of KYSE150R cells expressing wild-type p70S6K (OE-WT) or the Q347A mutant (OE-Q347A), followed by treatment with vehicle or CEP. All quantitative data are presented as mean ± SD from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant

Fig 4: CEP directly targets p70S6K, a master metabolic regulator and prognostic biomarker in ESCC. A Venn diagram illustrating the intersection of predicted CEP targets with glycolysis and oxidative phosphorylation (OXPHOS) gene sets. B Protein-protein interaction (PPI) network of the 39 overlapping core genes. C KEGG pathway enrichment analysis of the intersecting genes, prominently highlighting the PI3K/Akt and mTOR signaling pathways. D Circular plot showing Gene Ontology (GO) enrichment analysis of the overlapping targets. E Key sub-network clusters extracted from the overarching PPI network utilizing the MCODE algorithm. F Molecular docking model simulating the binding interface and affinity between CEP and the p70S6K kinase domain (interacting at the Gln347/Q347 residue). G Cellular thermal shift assay (CETSA) demonstrating that CEP binding enhances the thermal stability of p70S6K in intact cells. H Surface plasmon resonance (SPR) sensorgram confirming the direct binding affinity (KD = 1.94 µM) between CEP and purified p70S6K protein. I Analysis of TCGA data showing elevated p70S6K mRNA expression in ESCC tumor tissues (n = 152) compared to normal esophageal tissues (n = 11). J Kaplan-Meier curves for disease-free survival (DFS) in ESCC patients stratified by high (n = 21) versus low (n = 51) p70S6K expression levels. K Representative immunohistochemistry (IHC) images comparing p70S6K protein expression in paired cancerous and adjacent normal tissues (Scale bars, 50 μm). L Quantification of p70S6K levels in radiosensitive (PR, n = 31) versus radioresistant (SD, n = 31) ESCC patients