Journal: Open Biology

Article Title: AMPK phosphorylates NAMPT to regulate NAD + homeostasis under ionizing radiation

doi: 10.1098/rsob.220213

Figure Lengend Snippet: AMPK phosphorylates NAMPT S314 under ionizing radiation. ( b–d, f–k ) Immunoblotting analyses were performed using indicated antibodies. ( a ) HOK cells were pre-treated with 10 µM KU55933, 1 µM NU7441, 2 µM AZD6738, 20 µM PD98059, 20 µM SP600125 or 5 µM Compound C for 2 h, and cells were treated with 10 Gy ionizing radiation. Cellular NAMPT activity was measured 30 min after irradiation. ** p < 0.01; ns, not significant. ( b,c ) HOK and HUVEC cells were treated with 10 Gy ionizing radiation, and cells were harvested 15 min after irradiation. Immunoblots ( b ) and immunoprecipitations ( c ) were performed using indicated antibodies. IR, ionizing radiation; WCL, whole cell lysate. ( d ) Bacterially purified His/Flag-NAMPT protein was incubated with purified active AMPK proteins (His-AMPK α 1, untagged AMPK β 1 and untagged AMPK γ 1) in the presence or absence of Compound C and [γ- 32 P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. ( e ) Alignment analyses of NAMPT S314 or T304 was performed among indicated species. S314 and T304 were shown in red, and the residues matches AMPK phosphorylation consensus was shown in blue. ( f ) Bacterially purified WT His/Flag-NAMPT protein or indicated mutants were incubated with purified active AMPK proteins in the presence [γ- 32 P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. ( g ) HOK cells were treated with 10 Gy ionizing radiation. Immunoblots were performed using indicated antibodies in the presence or absence or NAMPT pS314 blocking peptide. ( h ) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation, and immunoprecipitation was performed 30 min after irradiation. ( i ) HOK cells with expression of Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and treated with 10 Gy ionizing radiation. Immunoprecipitation was performed 30 min after irradiation. ( j ) HOK cells were treated with 0.5 mM A769662 for 30 min. ( k ) HOK cells were incubated with glucose-free medium for 12 h. Glc, glucose.

Article Snippet: Antibodies recognizing NAMPT (#86634), ACC (#3676), ACC pS79 (#11818), AMPK α pT172 (#50081), and AMPK α (#5831) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Activity Assay, Irradiation, Purification, Incubation, In Vitro, Kinase Assay, Immunoprecipitation, Radioactivity, Autoradiography, Phospho-proteomics, Blocking Assay, Expressing

Journal: Open Biology

Article Title: AMPK phosphorylates NAMPT to regulate NAD + homeostasis under ionizing radiation

doi: 10.1098/rsob.220213

Figure Lengend Snippet: AMPK-dependent S314 phosphorylation activates NAMPT by facilitating NAMPT/PRPP association. ( a ) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the NAMPT enzymatic activity in the precipitates was measured. *** p < 0.001. ( b ) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the NAMPT enzymatic activity in the precipitates was measured. IR, ionizing radiation; WCL, whole cell lysate; ** p < 0.01. ( c ) The NAMPT catalytic domain in human NAMPT protein (shown in cyan) structure was boxed by dotted line, and enlarged to show the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple, PDB code: 2E5C), and nicotinamide (oxygen atom shown in purple, PDB code: 2E5D). ( d ) Human NAMPT protein structure (PDB code: 2E5C) shows the spatial location of S314 (side chain shown in red), PRPP (oxygen atom shown in purple), and H247 (side chain shown in orange). ( e ) Bacterially purified unheated WT His/Flag-NAMPT or S314 mutant protein was incubated with purified active AMPK proteins for an in vitro kinase assay. His/Flag-NAMPT proteins were precipitated using anti-Flag antibody, and the binding affinity between NAMPT protein and PRPP was measured. *** p < 0.001. ( f ) HOK cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS, and the binding affinity between NAMPT protein and PRPP was measured. ** p < 0.01. ( g ) HOK cells with expression of WT Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and cells were then treated with 10 Gy ionizing radiation. Flag-NAMPT proteins were precipitated from cell lysates 30 min after irradiation, washed twice with PBS and the binding affinity between NAMPT protein and PRPP was measured. ** p < 0.01.

Article Snippet: Antibodies recognizing NAMPT (#86634), ACC (#3676), ACC pS79 (#11818), AMPK α pT172 (#50081), and AMPK α (#5831) were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, Purification, Mutagenesis, Incubation, In Vitro, Kinase Assay, Activity Assay, Expressing, Irradiation, Binding Assay

Journal: Open Biology

Article Title: AMPK phosphorylates NAMPT to regulate NAD + homeostasis under ionizing radiation

doi: 10.1098/rsob.220213

Figure Lengend Snippet: A schematic of AMPK-mediated NAMPT activation under ionizing radiation. Ionizing radiation causes rapid activation of AMPK, which in turn phosphorylates NAMPT S314 and enhances NAMPT enzymatic activity. NAMPT activation promotes NAD + synthesis, thereby facilitating DNA repair and cell viability.

Article Snippet: Antibodies recognizing NAMPT (#86634), ACC (#3676), ACC pS79 (#11818), AMPK α pT172 (#50081), and AMPK α (#5831) were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Activity Assay

Journal: Clinical Cancer Research

Article Title: Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer

doi: 10.1158/1078-0432.ccr-21-4418

Figure Lengend Snippet: Figure 1. TERT elevates NADH and the NADH/NADþ ratio in cancer cells via upregulation of NAMPT and GAPDH. NADH (A), NADþ (B), and NADH/NADþ ratio (C) in TERTþ and TERT glioblastoma (GBM1, GBM6), oligodendroglioma (SF10417, BT88), melanoma (A375), hepatocellular carcinoma (HepG2), and neuroblastoma (SK-N-SH) cells. D, Schematic illustration of the link between TERT, FOXO1, NAMPT, GAPDH, NADH, and pyruvate flux to lactate in TERTþ cancer cells. NAM refers to nicotinamide; NMN is nicotinamide mononucleotide. NAMPT activity (E), GAPDH mRNA (F), and GAPDH activity (G) in TERTþ and TERT glioblastoma (GBM1, GBM6), oligodendroglioma (SF10417, BT88), melanoma (A375), hepatocellular carcinoma (HepG2), and neuroblastoma (SK-N-SH) cells. Bars depict meanvalues and error bars represent SD. , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: ForNAMPT and GAPDH overexpression in TERT cells, cells were transfected with siTERT siRNA for 24 hours, washed, and transiently transfected with human NAMPT or GAPDH cDNA (Origene), and expression confirmed by quantitative RT-PCR.

Techniques: Activity Assay

Journal: Clinical Cancer Research

Article Title: Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer

doi: 10.1158/1078-0432.ccr-21-4418

Figure Lengend Snippet: Figure 2. TERT acts via the FOXO1 transcription factor to upregulate NAMPT and GAPDH. A, Western blots for phosphorylated and total FOXO1 protein in TERTþ and TERT glioblastoma (GBM1), oligodendroglioma (BT88), melanoma (A375), neuroblastoma (SK-N-SH), and hepatocellular carcinoma (HepG2) cells. B, FOXO1 transcription factor activity in TERTþ and TERT glioblastoma (GBM1, GBM6), oligodendroglioma (SF10417, BT88), melanoma (A375), hepatocellular carcinoma (HepG2), and neuroblastoma (SK-N-SH) cells. Effect of expressing a constitutively active form of FOXO1 (CA-FOXO1) in TERTþ cells on NAMPT activity (C), GAPDH activity (D), NADH (E), and the NADH/NADþ ratio (F) in glioblastoma (GBM1, GBM6), oligodendroglioma (SF10417, BT88), melanoma (A375), hepatocellular carcinoma (HepG2), and neuroblastoma (SK-N-SH) models. Bars depict mean values and error bars represent SD. , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: ForNAMPT and GAPDH overexpression in TERT cells, cells were transfected with siTERT siRNA for 24 hours, washed, and transiently transfected with human NAMPT or GAPDH cDNA (Origene), and expression confirmed by quantitative RT-PCR.

Techniques: Western Blot, Activity Assay, Expressing

Journal: Clinical Cancer Research

Article Title: Deuterium Metabolic Imaging Reports on TERT Expression and Early Response to Therapy in Cancer

doi: 10.1158/1078-0432.ccr-21-4418

Figure Lengend Snippet: Figure 3. [U-2H]-pyruvate can noninvasively monitor TERT expression in vivo. FOXO1 transcription factor activity (A), NAMPT activity (B), and GAPDH activity (C) in glioblastoma, oligodendroglioma, and gliosis biopsies. D, Representative 2H-MR spectra from TERTþ and TERT BT88 cells cultured in medium containing 10 mmol/L [U-2H]-pyruvate for 72 hours. A representative spectrum acquired from neat culture medium containing 10 mmol/L [U-2H]-pyruvate is also shown. Peaks for semiheavy water (HDO; 4.75 ppm), pyruvate (2.4 ppm), and lactate (1.3 ppm) are labeled. E, Effect of TERT silencing on 2H-lactate production from [U-2H]- pyruvate in glioblastoma (GBM1), oligodendroglioma (BT88), melanoma (A375), hepatocellular carcinoma (HepG2), and neuroblastoma (SK-N-SH) cells. F, Representative 2H-MR spectral array acquired from a saline-treated mouse bearing a subcutaneous HepG2dox-TERT tumor following intravenous injection of [U-2H]-pyruvate. The first spectrum is acquired prior to [U-2H]-pyruvate injection. Inset shows an expansion of the spectrum with the highest lactate signal (second spectrum following [U-2H]-pyruvate injection). G, Concentration of 2H-lactate produced from [U-2H]-pyruvate at day 0 and day 7 in saline- and doxycycline (DOX)- treated mice bearing subcutaneous HepG2dox-TERT tumors. H, Tumor volume at day 0 and day 7 in saline- and doxycycline (DOX)-treated mice bearing subcutaneous HepG2dox-TERT tumors. Bars depict mean values and error bars represent SD. , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: ForNAMPT and GAPDH overexpression in TERT cells, cells were transfected with siTERT siRNA for 24 hours, washed, and transiently transfected with human NAMPT or GAPDH cDNA (Origene), and expression confirmed by quantitative RT-PCR.

Techniques: Expressing, In Vivo, Activity Assay, Cell Culture, Labeling, Saline, Injection, Concentration Assay, Produced

Journal: Antioxidants

Article Title: The Involvement of Sirtuin 1 Dysfunction in High-Fat Diet-Induced Vascular Dysfunction in Mice

doi: 10.3390/antiox11030541

Figure Lengend Snippet: Reduced NAD + level, NAD + /NADH ratio and NMAPT expression in PVAT of diet-induced obese mice. Male C57BL/6J mice were treated with NCD or HFD for 20 weeks. NAD + level and NADH in mouse PVAT lysate were measured with NAD + /NADH Colorimetric Assay kit ( A ). Columns represent mean ± SEM. * p < 0.05, ** p < 0.01, n = 6. The mRNA expression of NAMPT ( B ) was studied with qPCR. Columns represent mean ± SEM. ns, not significant; ** p < 0.01, n = 6. The protein expression of NAMPT ( C ) was studied with Western blot. The blots shown are representative for 3 independent experiments with similar results. ns, not significant; * p < 0.05; n = 10. Parp1 and Parp2 expression ( D ) was studied with (qPCR). n = 10. ns, not significant.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against the eNOS (BD Transduction Laboratories; Cat# 610297); rabbit polyclonal antibody against SIRT1 (LifeSpan BioSciences; Cat# LS-B1564), rabbit polyclonal antibody against NAMPT (Aviva Systems Biology, Cat# ARP42255-T100), and mouse monoclonal antibody against β-tubulin (Sigma-Aldrich, Cat# T7816).

Techniques: Expressing, Colorimetric Assay, Western Blot

Journal: Antioxidants

Article Title: The Involvement of Sirtuin 1 Dysfunction in High-Fat Diet-Induced Vascular Dysfunction in Mice

doi: 10.3390/antiox11030541

Figure Lengend Snippet: PVAT eNOS dysregulation in diet-induced obese mice. Diet-induced obesity leads to inhibition of Akt phosphorylation. The downregulation of NAMPT reduces cellular NAD + levels and SIRT1 activity. The resulting reduction in eNOS phosphorylation and enhancement of eNOS acetylation both decrease eNOS activity. In addition, HFD feeding causes eNOS uncoupling due to arginine deficiency because of arginase induction. The illustrated mechanisms are a summary of results from the present study and two previous publications [ , ].

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody against the eNOS (BD Transduction Laboratories; Cat# 610297); rabbit polyclonal antibody against SIRT1 (LifeSpan BioSciences; Cat# LS-B1564), rabbit polyclonal antibody against NAMPT (Aviva Systems Biology, Cat# ARP42255-T100), and mouse monoclonal antibody against β-tubulin (Sigma-Aldrich, Cat# T7816).

Techniques: Inhibition, Phospho-proteomics, Activity Assay

Journal: STAR Protocols

Article Title: Protocol to study inter-tissue communication between the hypothalamus and white adipose tissue and lifespan using a chemogenetic approach in aged mice

doi: 10.1016/j.xpro.2024.103551

Figure Lengend Snippet:

Article Snippet: Rabbit anti-NAMPT (1:1,000) , Bethyl , A700-058 RRID: AB_2891856.

Techniques: Plasmid Preparation, Virus, Recombinant, Saline, Ointment, Protease Inhibitor, Software, Microscopy, Membrane, Bicinchoninic Acid Protein Assay, Spectrophotometry, Western Blot, Stripping