Fig 1: Effects of OGT inhibition on cell viability and establishment of the dose for metabolomicsA. Enzymes regulating O-GlcNAc cycling. GFPT1 (glutamine--fructose-6-phosphate transaminase 1) is the rate-limiting enzyme in the hexosamine (HBP) biosynthetic pathway and directs glucose to the HBP rather than glycolysis. UDP-GlcNAc (UDP-N-acetylglucosamine) is the end-product of HBP and is utilized by OGT to modify target proteins via single sugar conjugation. c-MYC is highlighted here as one of its targets. ST045849 is a small molecule inhibitor targeting OGT. OGA (N-Acetyl-Beta-D-Glucosaminidase) removes O-GlcNAc from target proteins. B. LNCaP and PNT2 cells were treated with the indicated doses of OGT inhibitor ST045849 for 96 hours, and the viability was determined with the CellTiter-Glow® (CTG) assay. The data shown is an average of four biological replicates and Standard Error of the Mean (SEM) is shown. The significance was assessed with Student's t-test, ** < 0.01 and *** < 0.001. C. Growth rate of cells after indicated treatments. The data shown is an average of four biological replicates with SEM. The significance was assessed with Student's t-test as above, * < 0.05, ** < 0.01 and *** < 0.001, red stars indicate comparison between normal and 20µM ST045849 and blue stars indicate comparison between normal and 40µM ST045849. D. Total mRNA was collected from LNCaP cells treated with 20µM OGT inhibitor ST045849 for 12, 24, 48 and 96 hours, and analysed with RT-qPCR. OGT inhibitor treated samples were normalized to sample without treatment at 12 hours. The data shown is an average of at least three biological replicates with SEM. The significance was assessed with Student's t-test (* < 0.05, ** < 0.01 and *** < 0.001) by comparing untreated sample from the corresponding time point, and each colour corresponds to the transcript measurement highlighted with the same colour. E. Protein lysates were harvested at 12, 24, 48 and 96 hour time-points and blotted for the markers of interest. The data shown is representative of three biological replicates. Densitometry was used to quantitate the intensity of each band, AR, c-MYC and CDK1 intensities were normalized to loading control and un-treated sample from each time-point was set to 1. F. Potential correlation of CDK1 expression with biochemical recurrence in prostate cancer patients was assessed using cBioPortal for Cancer Genomics (http://www.cbioportal.org/) using Taylor & al. data set [25]. Increased expression of CDK1 predicts biochemical recurrence with p value of 0.00179.