Fig. 1: Differential phosphoproteomics between parental (HuH-7) cells and cells with acquired resistance to sorafenib (HuH-7^R).

a Workflow for quantitative phosphoproteomic analyses between parental (HuH-7) and HCC cells with acquired sorafenib resistance (HuH-7^R) via SILAC-based mass spectrometry. Heavy and light cell lysates were mixed and digested with trypsin and fractionated by high-pH reverse-phase chromatography. Phosphopeptides were then purified with TiO₂ column and analyzed in an LTQ-Orbitrap Velos hybrid mass spectrometer. b Clustered gene ontology functional enrichment was assessed with DAVID. Upregulated phosphoproteins in HuH-7^R cells showing a SILAC fold change H/L ≥ 1.5 were analyzed. The top three functional clusters are listed. −Log (p values) and enrichment scores are presented. c Pathway enrichment analysis of the upregulated phosphoproteins in HuH-7^R cells based on the KEGG pathway database with DAVID. Pathways with p values < 0.05 are shown. d Interaction linkage analysis of the molecules in enriched pathways shown in c with STRING. The connected molecules are shown. e Schematic representation of the postulated dysregulated phosphoprotein functional network in sorafenib-resistant HCC (HuH-7^R) cells. Gray, identified upregulated phosphoproteins; white, molecules that are postulated but not defined; black bold arrows, signaling linkages.