Extended Data Fig. 2: Transcriptomics and metabolomics analyses reveal NAT10 regulates serine metabolism in AML cells.
(a) The schematic diagram showing the targeting sites of NAT10 shRNAs. (b) mRNA levels of NAT10 in MOLM13 (left) and MonoMac6 (right) cells on day 4 after NAT10 knockdown. (c) Western blotting showing the knockdown efficiency of NAT10 in MonoMac6 cells on day 4 post-transduction of lentiviral shRNAs. (d) MTT assays showing the effects of NAT10 knockdown on cell proliferation in MonoMac6 cells. Cells were seeded on day 4 post-transduction (denoted as day 0 in the growth curves in MTT). (e) Histograms showing the percentages of apoptotic cells in control or NAT10 knockdown MOLM13 and MonoMac6 cells as detected by FITC-Annexin V/PI staining and flow cytometry analysis. Annexin V positive cells were defined as apoptotic cells. Apoptosis was detected on day 5 post-transduction of lentiviral shRNAs. (f) Flow-cytometric analysis of cell apoptosis in MOLM13 (upper) and MonoMac6 (bottom) cells on day 5 after NAT10 knockdown. (g) Gene set enrichment analysis (GSEA) of DEGs in MOLM13 cells after NAT10 knockdown. (h) Bubble diagram showing enrichment of metabolic pathways by the metabolites with reduced level after NAT10 knockdown in MOLM13 cells. (i) Heatmap showing the changes of detected amino acids on day 4 after NAT10 knockdown in MOLM13 cells. (j) Bubble diagram showing the most impacted metabolic pathways with enrichment of reduced metabolites upon NAT10 knockdown in MOLM13 cells. (k) Total levels of serine measured by LC–MS in MOLM13 cells transduced with NAT10 shRNAs or shNS for 4 days. (l) Total levels and isotopologue distribution (m + x; x, numbers of 13C) of GSH measured by LC–MS in NAT10 knockdown and control MOLM13 cells on day 4 post-transduction of shRNAs. Cells were grown in medium containing U-13C-glucose for 16 hours before sample collection. Values are mean ± s.d. of n = 3 biological replicates in b, d, e, k and l. Two-tailed Student’s t-tests were used in b, d, e, and k. Images in c and f were representative of three independent experiments.
