Extended Data Fig. 1: OGT regulates de novo nucleotide synthesis, not by affecting PRPS1 and PRPS2 protein levels.

a,b, The indicated siRNAs were introduced into A549 cells. Cells were incubated in medium containing ¹³C₆-glucose. LC/MS-MS analysis was performed to measure the levels of ¹³C₆ labeled nucleotide monophosphates (a) and PRPP (b). (n = 3). c, The wild-type, PRPS1 knockout, PRPS2 knockout, and double knockout A549 cells were incubated in the medium containing ¹³C₆-glucose. LC/MS-MS analysis was performed to measure the incorporation of ¹³C₆-glucose into intracellular PRPP . The proteins purified from the cells were subject to immunoblot analyses with the indicated antibodies. (n = 3). d, The indicated plasmids were introduced into H1299 cells. FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. Immunoblotting was conducted with indicated antibodies. The intensity of FLAG-PRPS1 immunoblots was used to quantify PRPS1 activity. (n = 3). e,f, The indicated shRNAs were introduced into HEK293T cells (n = 3) (e). Myc-vector or Myc-OGT plasmids were introduced into HEK293T cells (n = 3) (f). The cells from all groups were transfected with FLAG-PRPS1 plasmids. FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. The intensity of FLAG-PRPS1 immunoblots was used to quantify PRPS1 activity. g, The indicated siRNAs were introduced into H1299 cells. Immunoblotting was conducted with indicated antibodies. The relative protein expression were quantified. (n = 3). h,i, The FLAG-PRPS1 plasmids were introduced into HEK293T cells. The cells were cultured with or without glucose for 24 h (n = 3) (h) or treated with or without 50 µM OSMI-1 for 24 h (n = 4) (i). FLAG-PRPS1 was purified and subjected to PRPS1 enzymatic activity assay. Immunoblotting was conducted with indicated antibodies. Intensities of FLAG-PRPS1 immunoblots were used to quantify the PRPS1 activity. Each error bar represents mean±SEM. Statistical analysis was done using a two-tailed Student’s t-test.

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