Fig. 2: Cellular targets of artesunate in AML cells.

a General workflow of the chemical proteomics approach where the leukemic cells are treated with biotinylated ART (50 μM, 6 h) and streptavidin pull-down followed by mass spectrometry. b Pie chart illustrating the cellular component enrichment from gene ontology (GO) analysis of identified direct protein targets. c KEGG pathway enrichment analysis of metabolites downregulated by ART in the U937 cells. (x-axis; enrichment ratio of the KEGG pathway and the intensity of red indicates the p-value highest to lowest). Pathway analysis of the protein targets and a table of proteins with the highest overall coverage. ACC, acetyl-CoA carboxylase 1; PC, pyruvate carboxylase; ACADVL, very long-chain specific acyl-CoA dehydrogenase; HSP60, heat shock protein 60 kDa; VDAC1, voltage-dependent anion-selective channel protein 1; FASN, Fatty acid synthase N. % Cov, percent protein sequence coverage with the identified peptides. d Fold change (FC) in the acylcarnitines levels identified in the untargeted metabolomics of U937 cells treated with ART (5 μM) for 24 h. e Immunoblot of VLCAD in U937 cells transduced with scrambled control shRNA and shRNA against VLCAD. f Representative electron micrographs of shVLCAD and shCNTRL U937 cells. Scale bars represent 0.5 μm. g Viability of U937 shCNTRL and shVLCAD cells treated with ATO and ART for 48 h (cultured in complete media) (n = 4; ATO = 2 μM; ART = 5 μM). h Flow cytometry histograms illustrate the cellular expression levels of VLCAD in the U937 cells transduced with a plasmid encoding VLCAD cDNA and its respective control. (OE: Overexpression). i Viability of U937 CNTRL and VLCAD OE cells treated with ATO and ART for 48 h (cultured in complete media) (n = 3; ATO = 2 μM; ART = 5 μM). Data are presented as mean ± SEM. n.s., P > 0.05; *P < 0.05; ***P < 0.001, ***P < 0.0001, with a two-tailed unpaired t-test or one-way analysis of variance.