Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A . The nutritional sensing Hexosamine Biosynthetic Pathway (HBP) integrates availability and metabolism of glucose (fructose-6-phosphate, F6P), amino acids (glutamine), fatty acids (Acetyl-CoA) and nucleotides (UTP) in generating UDP-GlcNAc, a nucleotide sugar donor for protein O-GlcNAcylation. Feeding cells with GlcNAc increases UDP-GlcNAc biosynthesis without substantially changing cell metabolism. The enzyme O-GlcNAc transferase (OGT) catalyzes the addition of O-GlcNAc to serine or threonine residues of proteins, while O-GlcNAcase (OGA) removes this modification. OSMI-1 and ST060266 are small-molecule inhibitors of OGT, while PUGNAc inhibits OGA. B-C . Western blots confirming knockdown of OGT and OGA in cells stably expressing doxycycline-inducible shRNAmir (shOGT or shOGA) compared to scrambled control (shScr) after 48 hours of doxycycline (1 μg/ml) treatment. D. Western blot showing the effects of OGT and OGA knockdown on global O-GlcNAcylation. shScr, shOGT, and shOGA cells were treated with 1 μg/ml doxycycline for 48 hours. As a control, shScr cells were also treated overnight with 50 μM PUGNAc (an OGA inhibitor) to increase O-GlcNAcylation. For B-D, total cell lysates were analyzed, and β-actin was probed as a loading control. E . Compared to untargeted control (shScr), silencing OGT (shOGT) or OGA (shOGA) modulates the DSB repair defect in MCF7 cells after irradiation with or without PARP inhibitor. shRNAs were induced for 48 h with 1 μg/ml doxycycline, and cells treated for 1 h with 0 or 10 μM veliparib before 6 Gy irradiation. Neutral comet assays were performed 24 h after 6 Gy. Plots show the percentage of tail DNA for individual nuclei (circles). Similarly, silencing OGT or OGA modulates γH2AX foci persistence in MCF7 cells after irradiation with or without PARP inhibitor. F . Representative images demonstrating effects of shScr, shOGT, and shOGA at 24 h after 6 Gy ± 10 μM veliparib on persistence of γH2AX foci (pseudo-colored in red) with DNA counterstained using DAPI (blue), with overlays. G . Plots show the number of γH2AX foci per nucleus in individual cells as shown in F . Silencing OGT or OGA also modulates 53BP1 foci persistence in MCF7 cells after irradiation with or without PARP inhibitor. H . Effects of shScr, shOGT, and shOGA at 24 h after 6 Gy ± 10 μM veliparib on 53BP1 foci (pseudo-colored in green) with DAPI (blue). I . Plots show the number of 53BP1 foci per nucleus in individual cells as shown in H . For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: To assess knockout efficiency, PARP1 or EZH2 protein expression in the knockout pools was evaluated by Western Blot analysis using an anti-PARP1 antibody (Cell Signaling Technology; 9532T) or anti-EZH2 antibody (Abcam; ab191080).

Techniques: Modification, Western Blot, Knockdown, Stable Transfection, Expressing, Control, Irradiation

Journal: Journal of cell science

Article Title: Cancer metabolism in radiation sensitization: complementary roles of O-GlcNAc transferase (OGT) and PARP1

doi: 10.1242/jcs.264322

Figure Lengend Snippet: A , B . Western blot validation of PARP1 ( A ) and EZH2 ( B ) knockout (KO) pools. MCF7 cells were electroporated with CRISPR RNP formed with sets of three sgRNAs targeting the PARP1 or EZH2 initiation codons or three non-targeted guides used as WT control. C-E . MCF7 WT ( C ), PARP1-KO ( D ), and EZH2-KO ( E ) cells were incubated with 10 μM of BrdU for 24 h, followed by 1 h treatment before 6 Gy with 0 or 10 μM veliparib ± 50 μM PUGNAc. After 24 h, cells were fixed and stained with anti-BrdU under non-denaturing conditions (upper) or with anti-RPA (lower). F , G . Plots of BrdU ( F ) or RPA ( G ) foci per nucleus for cells treated as in C - E . For images, scale bar = 20 μm; inset is mean ± SEM foci per nucleus. For plots, red bars indicate mean ± SEM; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05 (unpaired t-test).

Article Snippet: To assess knockout efficiency, PARP1 or EZH2 protein expression in the knockout pools was evaluated by Western Blot analysis using an anti-PARP1 antibody (Cell Signaling Technology; 9532T) or anti-EZH2 antibody (Abcam; ab191080).

Techniques: Western Blot, Biomarker Discovery, Knock-Out, CRISPR, Control, Incubation, Staining