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. 2021 Jul 10;12(7):693. doi: 10.1038/s41419-021-03970-8

Fig. 5. NSLC01 inhibits the asparagine de novo synthesis pathway by targeting protein translation.

Fig. 5

A Experimental flow for the metabolic flux assay: BXPC3 (resistant to NSLC01) and ASPC1 (sensitive to NSLC01) were treated with NSC or DMSO for 24 h; cell medium was replaced with fresh medium supplemented with 13C-glucose, 15N2-glutamine, and NSLC01 (3 µM) for an additional 24 h; and analysis of amino acids was performed using liquid chromatography/high-resolution mass spectrometry. B, C The relative 13C (B) and 15N (C) enrichment ratios of metabolites with detectable 13C or 15N enrichment were calculated using the percentage of 13C-labeled or 15N-labeled metabolite in NSLC01-treated cells compared with those in control-treated cells. Metabolites without detectable 13C or 15N enrichment are not shown. D Western blot analysis of the key molecules in protein translation and, asparagine and serine synthesis pathways. E Protein synthesis assay using Click-It HPG kits: cells were treated with NSLC01 (3 µM) or control for 48 h, and nascent protein synthesis was analyzed with Click-It HPG Alexa Fluor protein synthesis assay kits. Red color indicates nascent synthesized proteins. F Quantifications of fluorescence inensity. G, H Immunohistochemistry staining of asparagine ASNS and PHGDH, respectively, in tumor specimens after treatment.