Extended Data Fig. 4: PRPS1 O-GlcNAcylation promotes tumor growth.

a, H1299 cells were treated with or without 50 µM OSMI-1 for 24h. Immunoblot analyses were conducted with indicated antibodies. (n = 3). b-e, The indicated plasmids were introduced into H1299 cells. The cells were subcutaneously injected into mice flank regions. Xenograft volumes were measured on the indicated days (b). By the end of the experiment, tumors were harvested and weighed after euthanization of mice (n = 5) (c). The lysate of homogenized xenografts was subjected to immunoprecipitation and immunoblotting with anti-FLAG agarose beads and indicated antibodies (d). FLAG-PRPS1 was purified from the xenograft lysates and subjected to PRPS1 enzymatic activity assays (n = 3) (e). f, The indicated PRPS1 plasmids were reintroduced in PRPS1-deleted H1299 cells. The cells were irradiated with a series of doses of radiation. The colony forming efficiencies were analyzed. (n = 3). g, The PRPS1 WT/2A plasmids were reintroduced in PRPS1-deleted H1299 cells. γ-H2AX focus positive cell ratios at the indicated time points after 2Gy irradiation were analyzed. (n = 3). h, The indicated PRPS1 plasmids were reintroduced in PRPS1-deleted H1299 cells. Cells were exposed to 50 µM etoposide for 2 h. Immunoblotting was conducted with indicated antibodies. (n = 2). i, H1299 cells were exposed to indicated doses of radiation. PRPS1 O-GlcNAcylation was analyzed using the chemoenzymatic labeling method. Immunoblot analyses were conducted with the indicated antibodies. (n = 3). j, Immunoblotting was conducted with indicated antibodies in H1299 parental and etoposide-resistant (Eto-R) cells. (n = 3). k, The indicated plasmids were introduced into HEK293T cells. Immunoprecipitation and immunoblot analyses were performed using anti-FLAG agarose beads and indicated antibodies. (n = 3). Each error bar represents mean±SEM. Two-tailed Student’s t-tests were employed to do statistical analysis.

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