free fatty acid quantification colorimetric  (Abcam)

Journal: Heliyon

Article Title: TRAF3IP2 drives mesenchymal stem cell senescence via regulation of NAMPT-mediated NAD biosynthesis

doi: 10.1016/j.heliyon.2023.e19505

Figure Lengend Snippet: TRAF3IP2 regulates MSC senescence via mediation of NAMPT–NAD biosynthesis. (A) Western blotting and quantitative analysis of NAMPT protein level in YMSCs and AMSCs. (B) Quantitative analysis of the intracellular NAD + concentration in YMSCs and AMSCs. (C) Representative images and quantitative analysis of SA-β-gal staining in control-lentivirus, TRAF3IP2-lentivirus or TRAF3IP2-lentivirus + P7C3-treated YMSCs. (D) Western blotting and quantitative analysis of NAMPT, p16 and p21 protein level in control-lentivirus, TRAF3IP2-lentivirus or TRAF3IP2-lentivirus + P7C3-treated YMSCs. (E) Quantitative analysis of the intracellular NAD + concentration in Control-lentivirus, TRAF3IP2-lentivirus or TRAF3IP2-lentivirus + P7C3-treated YMSCs. (F) Representative images and quantitative analysis of SA-β-gal staining in Control-siRNA, TRAF3IP2-siRNA or TRAF3IP2-siRNA + FK866-treated AMSCs. (G) Western blotting and quantitative analysis of NAMPT, p16 and p21 protein level in control-siRNA, TRAF3IP2-siRNA or TRAF3IP2-siRNA + FK866-treated AMSCs. (H) Quantitative analysis of the intracellular NAD + concentration in control-siRNA, TRAF3IP2-siRNA or TRAF3IP2-siRNA + FK866-treated AMSCs. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. **p < 0.01, ***p < 0.001. The raw data of A, D, G is presented in .

Article Snippet: Intracellular NAD + concentration of MSCs from different groups was measured using a NAD+/NADH Quantification Kit (Colorimetric) (ab65348; Abcam) as previously described [ ].

Techniques: Western Blot, Concentration Assay, Staining

Journal: Heliyon

Article Title: TRAF3IP2 drives mesenchymal stem cell senescence via regulation of NAMPT-mediated NAD biosynthesis

doi: 10.1016/j.heliyon.2023.e19505

Figure Lengend Snippet: TRAF3IP2 regulates NAMPT–NAD biosynthesis via the AMPK signaling pathway. (A) Western blotting and quantitative analysis of p-AMPK and AMPK protein level in YMSCs and AMSCs. (B) Western blotting and quantitative analysis of p-AMPK, AMPK and NAMPT protein level in Control-lentivirus, TRAF3IP2-lentivirus or TRAF3IP2-lentivirus + AICAR-treated YMSCs. (C) Quantitative analysis of the intracellular NAD + concentration in Control-lentivirus, TRAF3IP2-lentivirus or TRAF3IP2-lentivirus + AICAR-treated YMSCs. (D) Western blotting and quantitative analysis of p-AMPK, AMPK and NAMPT protein level in Control-siRNA, TRAF3IP2-siRNA or TRAF3IP2-siRNA + Compound C-treated AMSCs. (E) Quantitative analysis of the intracellular NAD + concentration in Control-siRNA, TRAF3IP2-siRNA or TRAF3IP2-siRNA + Compound C-treated AMSCs. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. **p < 0.01, ***p < 0.001. The raw data of A, B, D is presented in .

Article Snippet: Intracellular NAD + concentration of MSCs from different groups was measured using a NAD+/NADH Quantification Kit (Colorimetric) (ab65348; Abcam) as previously described [ ].

Techniques: Western Blot, Concentration Assay

Journal: Heliyon

Article Title: TRAF3IP2 drives mesenchymal stem cell senescence via regulation of NAMPT-mediated NAD biosynthesis

doi: 10.1016/j.heliyon.2023.e19505

Figure Lengend Snippet: TRAF3IP2 mediates the replicative senescence of MSCs. (A) Representative images and quantitative analysis of SA-β-gal staining in P3, P6 and P12 of YMSCs. (B) Western blotting and quantitative analysis of p-AMPK, AMPK and NAMPT protein level in P3, P6 and P12 of YMSCs. (C) Quantitative analysis of the intracellular NAD + concentration in P3, P6 and P12 of YMSCs. (D) Representative images and quantitative analysis of SA-β-gal staining in P12 of YMSCs with Control-siRNA, TRAF3IP2-siRNA, TRAF3IP2-siRNA + Compound C or TRAF3IP2-siRNA + FMK866 treatment. (E) Western blotting and quantitative analysis of p-AMPK, AMPK and NAMPT protein level in P12 of YMSCs with Control-siRNA, TRAF3IP2-siRNA, TRAF3IP2-siRNA + Compound C or TRAF3IP2-siRNA + FMK866 treatment. (F) Quantitative analysis of the intracellular NAD + concentration in P12 of YMSCswith Control-siRNA, TRAF3IP2-siRNA, TRAF3IP2-siRNA + Compound C or TRAF3IP2-siRNA + FMK866 treatment. (G) Concentration of TNF-α, IL-6 and IL-10 in CdM isolated from P12 of YMSCs with Control-siRNA, TRAF3IP2-siRNA, TRAF3IP2-siRNA + Compound C or TRAF3IP2-siRNA + FMK866 treatment. All data were obtained from at least three independent experiments and each error bar represents the mean ± SEM. **p < 0.01, ***p < 0.001. The raw data of B, E is presented in .

Article Snippet: Intracellular NAD + concentration of MSCs from different groups was measured using a NAD+/NADH Quantification Kit (Colorimetric) (ab65348; Abcam) as previously described [ ].

Techniques: Staining, Western Blot, Concentration Assay, Isolation

Journal: Nutrients

Article Title: Nicotinamide Prevents Diabetic Brain Inflammation via NAD+-Dependent Deacetylation Mechanisms

doi: 10.3390/nu15143083

Figure Lengend Snippet: Effect of NAM on the brain NAD+ content and acetylated form of NFκB (p65) in brains of STZ-induced diabetic mice. ( a ) Brain NAD+ content. ( b ) Relative protein abundance of the acetylated NFκB (p65) obtained by western blot analysis and representative images of brain acetylated NFκB (p65) in each condition. ( c ) Relative protein abundance of the acetylated NFκB (p65) obtained by IHC analysis and representative images of brain acetylated NFκB (p65) in each condition. Abbreviations used: NAD+, oxidized form of nicotinamide adenine dinucleotide; NAM LD, low-dose, NAM-treated, diabetic mice; NAM HD, high-dose, NAM-treated, diabetic mice; non-T1D, group of mice without T1D; T1D, group of mice with T1D. Data are expressed as the mean (standard deviation) of 4–5 mice/group. Images of 10 randomly selected fields were used for quantification. The scale bar shown in the images represents 100 µm. Statistically significant differences among groups for each variable were determined using a parametric ANOVA test followed by Tukey’s posttest. Differences were considered significant when p -value < 0.05. Specifically, * p -value < 0.05 vs. non-diabetic group; † p -value < 0.05 vs. diabetic group.

Article Snippet: NAD+ levels were determined in brain tissue using an NAD/NADH colorimetric determination kit (cat# ab65348 Abcam) according to the manufacturer’s instructions.

Techniques: Western Blot, Standard Deviation

Journal: Nutrients

Article Title: Nicotinamide Prevents Diabetic Brain Inflammation via NAD+-Dependent Deacetylation Mechanisms

doi: 10.3390/nu15143083

Figure Lengend Snippet: Relationship between relative NAD+ content and biomarkers of inflammation and microglial activation in brains from diabetic mice. ( a ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain NFκB (p65). ( b ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain acetyl-NFκB (p65). ( c ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain IBA-1. ( d ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain BDKRB-1. ( e ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain TNFα. ( f ) Correlation between relative levels of brain NAD+ content and IHC protein abundance of brain F4/80. The relationship between parameters was tested using a parametric Pearson’s correlation test. The relative levels of brain NAD+ were calculated taking as 1 the mean of individual values for this parameter in untreated diabetic mice. Mice of all groups were considered for analysis.

Article Snippet: NAD+ levels were determined in brain tissue using an NAD/NADH colorimetric determination kit (cat# ab65348 Abcam) according to the manufacturer’s instructions.

Techniques: Activation Assay

Journal: Communications Biology

Article Title: White spot syndrome virus (WSSV) modulates lipid metabolism in white shrimp

doi: 10.1038/s42003-023-04924-w

Figure Lengend Snippet: The indicated tissues of PBS-injected and WSSV-injected shrimp were collected at 12 hpi and 24 hpi and subjected to a , b lipase activity measurement and ( c ) free fatty acid quantification. Lipase activity was increased in infected hepatopancreas at 12 hpi. Free fatty acids were increased in hemocytes and hemolymph at 12 hpi, whereas a significant decrease of free fatty acids was found in infected hepatopancreas at 24 hpi. Each bar represents mean ± SD, n = 2 ~ 3 pool samples (3 shrimp per pool). Asterisks indicate differences between WSSV and PBS groups (* p < 0.05, ** p < 0.01). Hcy hemocytes, Hly hemolymph, Stm stomach, Hep hepatopancreas.

Article Snippet: A Free Fatty Acid Quantification Colorimetric/Fluorometric Kit (BioVision Inc.) was used to detect the unbound long-chain fatty acids in each shrimp sample.

Techniques: Injection, Activity Assay, Infection