Fig. 5: PRPS1 O-GlcNAcylation promotes tumor growth.
From: Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation
a,b, The indicated plasmids were reintroduced into PRPS1-deleted H1299 cells. Cells were labeled with 10 µM EdU for 30 min, and EdU-positive cells were examined by immunostaining. The quantification is shown in the middle panel. Protein levels were examined by western blot in the right panel (a) (n = 3). Cell growth was analyzed by measuring OD450 in cell viability assays. The box-and-whisker plots show all the points from the minimum to maximum values (b) (n = 5). c,d, H1299 cells were transfected with the indicated constructs and injected subcutaneously into flank regions of nude mice. Mice were treated with PBS or etoposide (20 mg kg−1, twice a week for 3 weeks), and xenograft volumes were measured on the indicated days (c). At the end of the experiment, mice were euthanized, and tumors were weighed. The xenografts were subjected to label-free LC–MS/MS analysis for nucleotide monophosphate levels (d) (n = 5). e, The indicated H1299 cells were seeded and treated with indicated concentrations of etoposide. The colony-forming efficiencies were analyzed (n = 3). f, The tumor weight ratios between control and etoposide-treated mice from c were calculated (n = 5). g, The indicated siRNAs were introduced into H1299 parental and Eto-R cells. Cells were exposed to indicated concentrations of etoposide for 48 h, and cell viability assays were performed (n = 4). The IC50 of etoposide was calculated for each group. h, The PRPS1 O-GlcNAcylation levels of H1299 parental and Eto-R cells were analyzed using the chemoenzymatic labeling method. Immunoblotting was conducted with the anti-PRPS1 antibody (n = 3). i, Endogenous PRPS1 purified from H1299 parental and Eto-R cells was subjected to PRPS1 enzymatic activity assays. Intensities of immunoblots were used to quantify PRPS1 activity (n = 4). j, Immunoprecipitation (IP) and immunoblotting were conducted with indicated antibodies in H1299 parental and Eto-R cells with indicated antibodies (n = 3). k, H1299 parental and Eto-R cells were incubated in the medium containing 13C6-glucose. LC–MS/MS was performed to measure the level of 13C6-labeled NAD (n = 3). Each error bar represents mean ± s.e.m unless indicated otherwise. A two-tailed Student’s t-test was employed for statistical analysis. conc., concentration.
