Fig. 6: Global proteomic, glycoproteomic, and phosphoproteomic analyses reveal the differentially regulated molecular features of arsenic MoA.

a The overview of MuPPE workflow for the dose-dependent treatment of NB4 cells with arsenic. Created in BioRender. Dong. (2025) [https://BioRender.com/b218mw3]. b Volcano plot depicting differentially expressed proteins upon arsenic treatment (10 μM vs 0 μM). c Volcano plot showing differentially regulated glycosylation sites following arsenic exposure (10 μM vs 0 μM). Inset: relative proportion of enzymes versus non-enzymes among glycoproteins with differential glycopeptide expression. d Volcano plot illustrating differentially regulated phosphorylation sites after arsenic treatment (10 μM vs 0 μM). Inset: percentage of kinases within proteins exhibiting differential phosphorylation. Downregulated entities are depicted in blue, upregulated in red, and unchanged in grey. e The counts of differentially regulated glycopeptides (left panel) and phosphopeptides (right panel), and total numbers of identified proteins, glycopeptides, and phosphopeptides across varying drug concentrations (middle panel). f Number of newly emerged or vanished glycopeptides and phosphopeptides compared with control group (0 μM). g Alterations in glycan structural profiles following arsenic treatment (10 μM vs 0 μM); HM = high-mannose glycans; comparison by two-sided P values (Student’s t-test), *P < 0.05, **P < 0.01. Error bars presented as mean ± SD, n = 4. h Differential regulation of kinases by family, as identified through KSEA. Cyan-shaded regions denote the proportion of regulated kinases within each family. i Schematic of PI3K-AKT-mTOR pathway perturbation by arsenic, with downstream effects on cell cycle and chromatin organization. Proteome (green), glycoproteome (orange), and phosphoproteome (blue) alterations are mapped. Created using ChemDraw (version 14.0). j Flow cytometry analysis of NB4 cells treated with increasing arsenic concentrations, showing dose-dependent G2/M phase accumulation comparison by two-sided P values (Student’s t-test), *P < 0.05, **P < 0.01. Error bars presented as mean ± SD, n = 3. k Differential expression of protein relative abundance, glycosylation sites, and phosphorylation sites of IGF2R protein with the log₂ fold change values. Different symbols representing upregulation (upward triangles), downregulation (downward triangles), and the color intensity indicating the significance of the change.