Journal: Oncogene

Article Title: O-GlcNAc modified-TIP60/KAT5 is required for PCK1 deficiency-induced HCC metastasis

doi: 10.1038/s41388-021-02058-z

Figure Lengend Snippet: a Interaction between endogenous OGT and KAT5 in MHCC-97H cells was detected by co-immunoprecipitation (co-IP) assay. b Subcellular co-localization of OGT and KAT5 in MHCC-97H cells were determined by immunofluorescence staining. Nuclei were counterstained with DAPI. Scale bar: 10 μm. c , d The interaction between HA-OGT and Flag-tagged full-length or truncated KAT5 (Chr 1-209aa, Zn 76-315aa, or HAT 233-513aa), as indicated in the diagram ( c ), were determined by co-IP in MHCC-97H cells ( d ). e Thiamet-G (TG) treatment enhances O-GlcNAc modification of KAT5. MHCC-97H cells were transfected with Flag-KAT5 or vector control for 48 h and treated with 25 μM TG for 12 h. Immunoprecipitation of Flag-KAT5 were performed with anti-FLAG antibody. f TG or ST045849 treatment regulates O-GlcNAc modification of KAT5. MHCC-97H cells were treated with 25 μM TG or 50 μM ST for 12 h, followed by succinylated wheat-germ agglutinin (sWGA) pull-down assay. Monosaccharide inhibitor GlcNAc (20 mM) was added as a negative control during sWGA-lectin-affinity purification. g , h PCK1-KO cells were treated with 50 μM ST for 12 h ( g ), MHCC-97H cells were transfected with vector control or HA-PCK1 for 48 h and treated with 25 μM TG for 12 h ( h ), followed by sWGA pull-down assay. i O-GlcNAc sites of KAT5 predicted using the YinOYang 1.2 server are shown with a black arrowhead at the top. The green vertical lines show the potential O-GlcNAc-modified Ser/Thr residues and the red horizontal wavy line indicates the threshold for modification potential. j Diagram of the potential O-GlcNAcylation sites on KAT5. k , l sWGA pull-down ( k ) or IP assay using anti-FLAG antibody ( l ). MHCC-97H cells were transfected with vector control, Flag-tagged WT, or mutants as indicated for 48 h, followed by sWGA pull-down or IP analysis using anti-FLAG antibody.

Article Snippet: Cells were cultured in medium supplemented with 6-diazo-5-oxo- l -norleucine (DON, 20 μM; D2141; Sigma-Aldrich, St Louis, MO, USA), ST045849 (ST, 50 μM; MFCD03308174; Tim Tec, Newark, USA), Thiamet G (TG, 25 μM; S7213; Selleckchem, Houston, USA) or Cycloheximide (CHX, 100 μM; HY-12320; MedChemExpress, New Jersey, USA) and then collected for analysis.

Techniques: Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Modification, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Pull Down Assay, Negative Control, Affinity Purification

Journal: Oncotarget

Article Title: Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

doi: 10.18632/oncotarget.7039

Figure Lengend Snippet: A. 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 . Increased expression of CDK1 predicts biochemical recurrence with p value of 0.00179.

Article Snippet: For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO).

Techniques: Conjugation Assay, CTG Assay, Comparison, Quantitative RT-PCR, Control, Expressing

Journal: Oncotarget

Article Title: Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

doi: 10.18632/oncotarget.7039

Figure Lengend Snippet: A. LNCaP cells were treated with 20μM OGT inhibitor ST045849 for 96 hours, cell media were collected and analyzed by 1 H NMR. An example of the obtained NMR spectra is shown. The levels of glucose and lactate were determined from the cell culture media by 1 H NMR. The data shown is an average of seven biological replicates with SEM. The significance was assessed with Student's t -test, ** < 0.01, *** < 0.001. B. Cells were treated as indicated in the figure and the viability of cells was analysed with CTG reagent after 96 hours treatment. Viability of the untreated sample was set to 100% and treatments were normalized to this. The data shown is an average of four biological replicates with SEM. The significance was assessed with Student's t -test, ** < 0.01, *** < 0.001. C. and D. Cells were treated as indicated in the figure and the growth rate of cells was recorded by life cell imaging. The data shown is an average of four biological replicates with SEM. The significance was assessed with Student's t -test, * < 0.05 and ** < 0.01. Black stars indicate comparison between rotenone (or metformin) only and combination of ST045849 with rotenone (or metformin), while red stars indicate comparison between ST045849 and combinatorial treatments.

Article Snippet: For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO).

Techniques: Cell Culture, Imaging, Comparison

Journal: Oncotarget

Article Title: Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

doi: 10.18632/oncotarget.7039

Figure Lengend Snippet: A. LNCaP cells were treated with OGT inhibitor ST045849 for 96 hours, cell lysates were collected and analysed by 1 H NMR. An example of the obtained NMR spectra is shown. Quantitation of the 1 H NMR data. The data shown is an average of seven biological replicates with SEM. The significance was assessed with Student's t -test, * < 0.05. B. Enzymatic reaction catalysed by GPT2 (glutamic pyruvate transaminase). C. Total mRNA was collected from cells treated with OGT inhibitor ST045849 for 12, 24, 48 and 96 hours, and analysed with RT-qPCR. OGT inhibitor ST045849 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 and significance was assessed by comparing untreated sample from the corresponding time point, * < 0.05, ** < 0.01. D. Glutamic pyruvate transaminase (GPT2) assay performed from cell lysates treated either with a vehicle or with 20μM of OGT inhibitor ST045849. The GPT2 activity was assessed at 48 hours. The data shown is an average of four biological replicates with SEM. The significance was assessed with Student's t -test, * < 0.05.

Article Snippet: For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO).

Techniques: Quantitation Assay, Quantitative RT-PCR, Activity Assay

Journal: Oncotarget

Article Title: Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

doi: 10.18632/oncotarget.7039

Figure Lengend Snippet: A. , B. and C. Cells were treated as indicated in the figure and the viability of cells was assessed after treatment with OGT inhibitor ST045849 or OSMI-1 alone or in combination with glutamic pyruvate transaminase (GPT2) inhibitors Cl-alanine or cycloserine. The data shown is an average of four biological replicates with SEM and significance was assessed with Student's t -test, * < 0.05, ** < 0.01 and *** < 0.001.

Article Snippet: For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO).

Techniques:

Journal: Oncotarget

Article Title: Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism

doi: 10.18632/oncotarget.7039

Figure Lengend Snippet: A. and B. Activation of caspases 3 and 7 was assessed in real time, imaging every 12 hours. The data shown is an average of four biological replicates with SEM and significance was assessed with Student's t -test, * < 0.05 and ** < 0.01. Red stars indicate comparison between either of the OGT inhibitors (ST045849 or OSMI-1) combined with Cl-alanine against any of the single treatments, while green stars indicate comparison between either of the OGT inhibitors (ST045849 or OSMI-1) combined with cycloserine against any of the single treatments.

Article Snippet: For metabolomic profiling, cells were plated into either media with a commercial OGT inhibitor (ST045849, TimTec) or a vehicle control (DMSO).

Techniques: Activation Assay, Imaging, Comparison