Journal: Cells

Article Title: Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity

doi: 10.3390/cells9102324

Figure Lengend Snippet: The reduction of OGA activity contributes to enhanced protein O-GlcNAcylation in NDPK-B-depleted ECs. ( A ) HUVECs transfected with either scrambled control (−) or siRNA against NDPK-B (siNDPK-B, +) were analyzed for OGT, OGA, and NDPK-B content by immunoblotting (n = 9). ( B ) Quantification of OGT and ( C ) OGA relative to loading control, γ-tubulin. ( D ) mRNA levels of OGT and ( E ) OGA relative to 18S quantified by qPCR (n = 6). OGA activity in ( F ) WT and KO retinae isolated from 5-month-old male mice (n = 8 mice per genotype), ( G ) lysates of control and NDPK-B knockdown HUVECs (n = 7) and ( H ) lysates of WT and KO MBMECs isolated from 10–15-week-old WT and KO mice. ( I ) NDPK-B and protein O-GlcNAc content analyzed by immunoblot in the same lysates of WT and KO MBMECs. ( J ) Quantification of protein O-GlcNAcylation relative to γ-tubulin (n = 5). MBMECs were prepared from 3–4 animals per genotype. WT: wild type; KO: NDPK-B knockout; OGT: O-GlcNAc transferase; OGA: O-GlcNAcase; MBMECs: murine brain microvascular ECs. Results are presented as mean ± SD. *: p < 0.05.

Article Snippet: The secondary antibodies used were rabbit anti-mouse peroxidase (A9044, Sigma-Aldrich, Munich, Germany, 1:20,000), goat-anti-rabbit peroxidase (A9169, Sigma-Aldrich, Munich, Germany, 1:40,000), and donkey-anti-sheep peroxidase (Sigma-Aldrich). qPCR primers were obtained from Applied Biosystems, ThermoFischer: GFAT Hs00899865_m1, OGA Hs00201970_m1, OGT Hs00269228_m1, and 18S Hs03003631_g1.

Techniques: Activity Assay, Transfection, Control, Western Blot, Isolation, Knockdown, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: O‐GlcNAcylation promotes malignancy and cisplatin resistance of lung cancer by stabilising NRF2

doi: 10.1002/ctm2.70037

Figure Lengend Snippet: H 2 O 2 increases NRF2 O‐GlcNAcylation through AMPK/OGT axis . (A) Interactions between OGT and NRF2 were determined by Co‐IP assay using NCI‐H1299 cells treated with H 2 O 2 (50 µM) for 45 min. Data represent three independent experiments. (B) NCI‐H1299 cells were stimulated with H 2 O 2 (50 µM) for 45 min and O‐GlcNAcylation of NRF2 was detected by chemoenzymatic labelling and WB analysis. Data represent three independent experiments. (C) Relative amounts of NRF2 in the nuclear or cytoplasmic fractions were determined by a cell fraction assay after treatment with H 2 O 2 (50 µM) for 45 min. Data represent three independent experiments. (D) ARE‐luc activity was determined by luciferase reporter assay in NCI‐H1299 NRF2‐WT and NCI‐H1299 NRF2‐S103A cells with a similar treatment in panel C (data represent three independent experiments, *** p < .001, ns, not significant, using one‐way ANOVA followed by Tukey's multiple comparisons test). (E) The interactions between NRF2 and OGT or KEAP1 were determined by Co‐IP assay in NCI‐H1299 cells treated with AICAR (1 mM) or A‐769662 (50 µM) for 1 h. Data represent three independent experiments. (F) O‐GlcNAcylation of NRF2 was detected by chemoenzymatic labelling and WB analysis in NCI‐H1299 cells treated with AICAR (1 mM) or A‐769662 (50 µM) for 1 h. Data represent three independent experiments. (G) ROS levels, as indicated by DCFH‐DA fluorescence, were measured by microplate reader in NCI‐H1299 cells, which were treated with AICAR (1 mM) or A‐769662 (50 µM) for 1 h (data represent three independent experiments, *** p < .001, using one‐way ANOVA followed by Dunnett's multiple comparisons test). (H) Representative images of p‐OGT (Thr444) and OGT proteins determined by WB in the presence of H 2 O 2 (50 µM) or AICAR (1 mM) for the indicated periods. Data represent three independent experiments. (I) The interaction between OGT and NRF2 was determined by Co‐IP assay after translating Flag‐tagged OGT WT , OGT T444E, or OGT T444A in NCI‐H1299 cells. Data represent two independent experiments. (J) Direct interaction of NRF2 with OGT WT and OGT T444E in an in vitro GST pull‐down assay. Data represent two independent experiments. The relative intensities of proteins in immunoblotting were determined by normalising the intensities of corresponding proteins to the intensities of β‐actin.

Article Snippet: The Anti‐Phospho‐OGT (Thr444) antibody was customised by ImmunoWay, as previously reported.

Techniques: Co-Immunoprecipitation Assay, Activity Assay, Luciferase, Reporter Assay, Fluorescence, In Vitro, Pull Down Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: O‐GlcNAcylation promotes malignancy and cisplatin resistance of lung cancer by stabilising NRF2

doi: 10.1002/ctm2.70037

Figure Lengend Snippet: Working model for regulation of NRF2 activity by O‐GlcNAcylation in cancer cells . Under normal circumstances, NRF2 is bound to KEAP1, which is a redox‐sensitive E3 ubiquitin ligase substrate adaptor molecule that regulates the ubiquitination and degradation of NRF2 (left). Upon oxidative stress or chemotherapy, AMPK is activated to phosphorylate OGT at the Thr444 site. The activated OGT is conducive to binding with NRF2 to increase the O‐GlcNAcylation of NRF2, which inhibits the binding of KEAP1 to NRF2 and thus promotes its stability, nuclear localisation, and transcription of its target genes, thus driving lung cancer cell proliferation, survival, and drug resistance.

Article Snippet: The Anti‐Phospho‐OGT (Thr444) antibody was customised by ImmunoWay, as previously reported.

Techniques: Activity Assay, Binding Assay