recombinant murine fgf21 rmfgf21  (Cusabio)

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a An overview of a general workflow for the study. b Comparison of pre- and post-transplantation Fgf21 mRNA expression in the liver graft based on the GSE15480 (n = 12 pairs, including pre- and post-LT donor livers) and GSE151648 (n = 40 pairs, including pre- and post-LT donor livers) derived from graft biopsies in liver transplantation. c Comparison of pre- and post-reperfusion FGF21 levels in the serum (Cohort 1, n = 88 pairs, including pre- and 2 h post-LT recipient serum). d The peripheral FGF21 level 2 h after reperfusion linearly correlated with maximal ALT within 7 days after transplantation. e The 88 patients were divided into the FGF21-elevated group (n = 44) and non-elevated group (n = 44) according to the median value of ratio change (post-reperfusion/pre-transplant). The elevated group had improved graft survival. f , g Pre-transplant FGF21 expression in biopsies (Cohort 2, n = 115) and its correlation with graft survival (after excluding liver grafts with CIT > 15 h, n = 107). h Information on graft steatotic change was missing for 19 cases in this cohort. We confirmed that 33 patients received steatotic liver grafts. Low FGF21 in the graft was associated with elevated ALT and AST after transplantation. However, there was no significant difference in the non-steatotic subgroup (n = 55). I/R, ischemia/reperfusion; KO knockout, ALT alanine aminotransferase, AST aspartate aminotransferase, CIT cold ischemia time. p values are shown on the graphs. Source data are provided as a file. Figure 1a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Comparison, Transplantation Assay, Expressing, Derivative Assay, Knock-Out

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a The time-dependent western blot analysis (left) and quantification (right) of FGF21 protein expression in the mouse liver undergoing ischemia for 1.5 h followed by the indicated duration of reperfusion, three independent biological mice samples. b Schematic for the establishment of the mouse I/R injury model. c Serum ALT and AST levels of wild-type and Fgf21 KO mice in the sham and I/R groups 6 h after reperfusion (n = 5, per group). d Representative H&E staining of liver sections from wild-type and Fgf21 KO mice in the sham and I/R groups. e Liver damage was evaluated using Suzuki’s histological score (n = 5, per group). f Representative TUNEL immunofluorescent staining in liver lobes of wild-type and Fgf21 KO mice in the sham and I/R groups. g Quantification analysis of the TUNEL-positive cells/high-power field (n = 5, per group). h Volcano plot of the differentially expressed genes (DEGs) between wild-type and Fgf21 KO livers after I/R treatment (n = 4) using the absolute value of log2 (fold change) >1.5 and p < 0.05 as the thresholds. i KEGG pathway enrichment analysis of the identified DEGs. j Heatmap of major metabolites catalyzed by lipoxygenases in wild-type and Fgf21 KO livers after I/R treatment (n = 4). The 15-HETE content was elevated in Fgf21 KO livers. k Co-expression network and KEGG pathway analysis of the hub metabolites. l The Pearson correlation coefficients between ALOX15/15-HETE (RNA-seq/metabolomic) and the co-expression modules identified by WGCNA. KEGG analysis of the module with the highest correlation coefficients ( p < 0.05 by Fisher’s exact test, two-sided). WT, wild-type, KO knockout, I/R ischemia/reperfusion, ALT alanine aminotransferase, AST aspartate aminotransferase. a , c , e , g , j Two-tailed t test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 2b created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Western Blot, Expressing, Staining, TUNEL Assay, RNA Sequencing, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a Wild-type and Fgf21 KO donor livers in the mouse orthotopic liver transplantation model were harvested, stored for 30 min at 4 °C, and transplanted into wild-type recipient mice. b Serum ALT and AST levels for each group after transplantation (n = 4, per group). c The 15-HETE level in the livers of each group 6 h after reperfusion (n = 4, per group). d The FGF21 level in the serum of each group 6 h after reperfusion (n = 4, per group). e Representative TUNEL staining of liver sections from each group (n = 4, per group). f Heatmap showing the differential expression of 41 immune markers in 34 cell clusters. g Two-dimensional t-SNE illustration of the CyTOF data of livers isolated from WT >> WT and KO >> WT (n = 4/group), the merged t-SNE graph was used for contrast. h – j The frequencies of the identified clusters in granulocytes, Mono/Mac cells and NK cells and the comparisons between the groups (n = 4, per group), boxplot shows the median (center line), 25th, and 75th percentile (lower and upper boundary), and the whiskers are essentially range bars that extend to max and min values. k Multiplex immunohistochemistry staining of NK1.1 (green), CD62L (red) and DAPI (blue) in Fgf21 KO-transplanted livers, three independent biological mice samples. Scale bar, 50 μm. OLT orthotopic liver transplantation, WT wild-type, KO knockout, ALT, alanine aminotransferase, AST aspartate aminotransferase, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling, Mono/Mac monocyte/macrophage, NK natural killer. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 4a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Transplantation Assay, TUNEL Assay, Staining, Quantitative Proteomics, Isolation, Multiplex Assay, Immunohistochemistry, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a Experimental schema for establishing the I/R injury mouse model. The mice were intravenously administered rmFGF21 (0.5 mg/kg). b Serum ALT and AST levels 6 h after reperfusion (n = 5, per group). c Comparison of serum FGF21 levels 6 h after reperfusion (n = 5, per group). d H&E staining of liver sections 6 h after reperfusion (n = 5, per group). e Western blot analysis (left) and quantification (right) of FGF21and BAX in AML12 from each group. f Western blot analysis (left) and quantification (right) of ALOX15, BAX and p-ERK1/2 in AML12 from each group. g Western blot analysis (up) and quantification (down) of ALOX15, BAX and p-FGFR4 in AML12 from each group. e – g three independent biological mice samples. I/R ischemia/reperfusion, ALT alanine aminotransferase, AST aspartate aminotransferase, H/R hypoxia/ reoxygenation. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 5a created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Comparison, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a IHC staining for FGF21 in pre-transplant biopsies from the steatotic liver grafts in Cohort 3 (n = 78). b Comparison of the degree of hepatic steatosis between the low FGF21 group and the high FGF21 group. c Comparison of maximal serum ALT/AST within 7 days after transplantation between the low FGF21 group and the high FGF21 group. d Comparison of FGF21 staining (mean integrated optical density, IOD) between patients who did and did not develop EAD. e Schematic for the establishment of the I/R injury model using mice fed a high-fat diet (HFD) or normal chow diet. After 8 weeks of feeding, mice were subjected to I/R treatment. f Serum ALT and AST 6 hours after reperfusion (n = 5, per group). g 15-HETE content in the livers from each group 6 h after reperfusion (n = 5, per group). h H&E staining, ROS staining and TUNEL staining of liver sections from each group 6 h after reperfusion (n = 5, per group). i Quantification assessment of infiltrating CD11b + and MPO + cells in the livers from each group (n = 5, per group). I/R ischemia/reperfusion, WT wild-type, KO knockout, HFD high-fat diet, ALT alanine aminotransferase, AST aspartate aminotransferase, HE hematoxylin and eosin, ROS reactive oxygen species, DHE dihydroethidium, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, MPO cytosolic myeloperoxidase. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, ns, not significant, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 6e created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Immunohistochemistry, Comparison, Transplantation Assay, Staining, TUNEL Assay, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: FGF21 modulates immunometabolic homeostasis via the ALOX15/15-HETE axis in early liver graft injury

doi: 10.1038/s41467-024-52379-2

Figure Lengend Snippet: a The steatotic mouse models underwent intravenous injection of rmFGF21 (0.5 mg/kg) or tail vein injection of AAV8-hAAT- Fgf21 before I/R injury. b 15-HETE content in the livers from each group 6 h after reperfusion (n = 4, per group). c Serum ALT and AST 6 h after reperfusion (n = 5, per group). d H&E staining, ROS staining and TUNEL staining and quantification results of liver sections from each group 6 h after reperfusion (n = 5, per group). e Quantification assessment of infiltrating CD11b + and MPO + cells in the livers of each group (n = 5, per group). f The general schematic diagram. LT, liver transplantation; AA, arachidonic acid. I/R, ischemia/reperfusion; HFD, high-fat diet; ALT, alanine aminotransferase; AST, aspartate aminotransferase; HE, hematoxylin and eosin; ROS, reactive oxygen species; DHE, dihydroethidium; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; MPO, cytosolic myeloperoxidase. Two-tailed t-test. Statistic data are presented as the mean ± SD, error bars represent the means of at least three independent experiments. p values are shown on the graphs, p < 0.05 was considered statistically significant. Source data are provided as a file. Figure 8a, f created with BioRender. com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ).

Article Snippet: Mice in the treatment groups were injected 1.5 h prior to the initiation of hepatic ischemia with a single dose of recombinant murine FGF21 (rmFGF21, 0.5 mg/kg i.v., C04D, Novoprotein), and HFD-fed mice were treated with rmFGF21 (0.5 mg/kg/d, i.p.) for 2 weeks prior to I/R.

Techniques: Injection, Staining, TUNEL Assay, Transplantation Assay, Two Tailed Test

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 1. PCB exposure induces NAFLD/NASH in mice fed either a SD and a HFD. Eight-week-old male C57BL/6 mice were fed either a SD or HFD for four weeks and then treated with vehicle (corn oil), Aroclor1260 (20 mg/kg) or PCB126 (5 mg/kg) by intraperitoneal injection for a total of four injections (two, three, four, and five weeks) during the six-week study duration. (A) Experimental design. (B) Representative images of H&E staining of liver tissue. Scale bar: 50 µm. (C) Quantification of the lipid droplet area in H&E-stained liver tissue. The data are expressed as a percentage of the area of lipid droplets in the field. (D) Hepatic TG. (E) Hepatic FFA. (F) Plasma FGF21. (G) ORO staining showing that both Aroclor1260 (20 µM) and PCB126 (10 µM) accelerated hepatic lipid accumulation in O/P-treated human ARE primary hepatocytes. Scale bar: 50 µm. (H) Representative images of H&E staining of adipose tissue. The crown-like structure is illustrated by the asterisk. Scale bar: 50 µm. (I) Average adipocyte size of eWAT was measured in H&E images using ImageJ 1.53 s. (J) Quantitative analysis of CLS formation in adipose tissue. All values represent the mean ± SD, n = 7–10 mice per group. * p < 0.05 and ** p < 0.01.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Injection, Staining, Clinical Proteomics

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 4. Recombinant FGF21 improves hepatic steatosis in PCB-induced NAFLD/NASH models. Aroclor1260- or PCB126-injected mice fed either a SD or HFD were intraperitoneally administered vehicle or rmFGF21 (1 mg/kg/day) once daily for 10 days. (A,B) Representative photograph images. (C,D) Liver weight. (E) H,E staining of liver tissue. Scale bar: 50 µm. (F) ORO staining and quantification showing that rhFGF21 (50 ng/mL, 24 h) reduced the hepatic lipid accumulation induced by both 20 µM Aroclor1260 and 10 µM PCB126 in O/P-treated human ARE primary hepatocytes. Scale bar: 50 µm. All values represent the mean ± SD, n = 4. * p < 0.05, ** p < 0.01 compared with the experimental control.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Injection, Staining, Control

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 5. Recombinant FGF21 attenuates hepatic iron overload in PCB-induced NAFLD/NASH models. Aroclor1260 (20 mg/kg)- or PCB126 (5 mg/kg)-injected mice fed either a SD or HFD were intraperitoneally administered vehicle or rmFGF21 (1 mg/kg/day) once daily for 10 days. (A) Prussian blue staining showing marked iron accumulation in liver samples. Scale bar: 50 µm. (B) Hepatic iron levels in PCB-induced NAFLD/NASH mice that were intraperitoneally administered vehicle or rmFGF21 (left panel, ferrous ion level; right panel, ferrous and ferric iron total levels). All values represent the mean ± SD, n = 7–10 mice per group. * p < 0.05 and ** p < 0.01. (C) Detection of intracellular ferrous ions (Fe2+) using the fluorescent probe FerroFarRed in O/P- and FAC-cotreated HepG2 cells. Scale bar: 200 µm.

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Injection, Staining

Journal: International journal of molecular sciences

Article Title: Recombinant FGF21 Attenuates Polychlorinated Biphenyl-Induced NAFLD/NASH by Modulating Hepatic Lipocalin-2 Expression.

doi: 10.3390/ijms23168899

Figure Lengend Snippet: Figure 7. Recombinant FGF21 reduces PCB-induced overexpression of hepatic LCN2. Ten days after the rmFGF21 administration (1 mg/kg/day) in Aroclor1260- or PCB126-induced NAFLD/NASH mice, (A) real-time PCR and (B) western blot analysis were performed to detect mouse LCN2 in liver tissues from mice fed either a SD or HFD. The values represent the mean ± SD, n = six to eight mice per group. * p < 0.05 and ** p < 0.01. (C) Western blot analysis was performed to assess human LCN2 in 20 µM Aroclor1260- or 10 µM PCB126-treated HepG2 cells cotreated with either vehicle or rhFGF21 (50 ng/mL, 24 h).

Article Snippet: Recombinant human FGF21 (rhFGF21) was purchased from Peprotech (Rocky Hill, NJ, USA), and recombinant murine FGF21 (rmFGF21) was purchased from Cusabio (Houston, TX, USA).

Techniques: Recombinant, Over Expression, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Browning marker gene quantification by qPCR in epididymal white adipose tissue (WAT) from WT mice fasted 0–48 h. n = 6 mice for each group. Circles, 0 h fast; squares, 12 h fast; upward triangle, 24 h; downward triangle, 48 h. B NAMPT, SIRT1, and FGF21 gene expression in livers from WT mice fasted 0–48 h. n = 6 mice for each group. Symbols: Circles, fasting 0 h. Squares, 12 h. Upward triangles, 24 h, downward triangle, 48 h. C Representative (from n = 4 per group) NAMPT immunoblot in livers from WT mice fasted 0–48 h. D qPCR quantification of NAMPT, SIRT1, FGF21 gene expression in primary hepatocytes treated with the fasting-mimetic glucose transporter inhibitors, trehalose and lactotrehalose. n = 4 per group. Circles, cultures treated with regular growth media, Red triangles, trehalose-treated, Blue squares, LactoTrehalose-treated. E qPCR in liver from WT mice treated with 5-day 3% trehalose ad libitum in drinking water. n = 8 vehicle- (black circles) and 18 trehalose-treated (red squares). F Fasting-inducible NAMPT expression by qPCR in isolated primary hepatocytes treated with or without 10 mM fructose in the presence or absence of carbohydrate transporter inhibitors trehalose or lactotrehalose. n = 3 starved, 4 fructose-treated, 4 trehalose- and fructose-treated, 4 lactotrehalose- and fructose-treated. Circles, cultures treated with regular growth media; squares, fructose-treated; downward triangles, cultures treated with trehalose and fructose; open circles, LactoTrehalose and fructose-treated cultures. Error bars in ( A – B ), ( D – F ) represent standard error of the mean (SEM). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Statistical tests: ( A – F ), two-tailed T -test, Bonferroni–Dunn post hoc.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Marker, Expressing, Western Blot, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Heat-Zeitgeber Time tracing in WT and NAMPT LKO littermate mice during light and dark cycles during fasting. Mean heat production in WT and NAMPT LKO mice during light and dark cycle fed and early (Fasting I, 1- 24 h) and prolonged (Fasting II, 24–48 h) fasting periods is graphed below. n = 3 WT, 4 NAMPT LKO mice. B RER-Zeitgeber time tracing in WT and NAMPT LKO littermate mice during light and dark cycles during ad-lib fed (first 24 h) and fasting periods. Mean RER in WT and NAMPT LKO mice during light and dark cycle fed and fasting periods is graphed below. n = 3 WT, 4 NAMPT LKO mice. Error bars in ( A – B ) (bottom panels) represent SEM. Circles, WT mice. Squares, NAMPT LKO mice for ( A and B ). C Liver triglycerides (TG), total cholesterol and non-esterified fatty acid (FFA) quantification following 48 h fasting in WT and NAMPT LKO mice. n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. D qPCR quantification of liver FGF21, NAMPT in fed and 48 h-fasting WT and NAMPT LKO mice. n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. Error bars in ( C and D ). represent SEM. Black circles, Fed WT, Red circles, fasting WT, Blue circles, Fasting NAMPT LKO mice. E Venn diagram representing significantly altered genes ( P < 0.05) in random-fed vs. 12 h fasting WT and NAMPT LKO mice, quantified by unbiased transcriptomics. n = 2 WT Fed, 4 WT Fasting; 5 NAMPT LKO Fasting. F Bar plot demonstrating −log(FC) for significantly down-regulated gene ontology (GO) pathways revealed when comparing WT vs. NAMPT LKO livers ( P < 0.05). n = 4 WT Fed, 6 WT Fasting; 4 NAMPT LKO Fasting. Statistical tests: ( A ) (upper panel), ANCOVA. A (lower panels), ( B – D ), two-tailed T -test, Bonferroni–Dunn post hoc correction. E , F EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Experimental design for db/db AdNAMPT analyses. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. B Body weight over time in db/db mice expressing GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. C Body composition by echoMRI analysis in mice expressing GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. D Serum triglycerides (TG), cholesterol and low-density lipoprotein cholesterol (LDL-C) in db/db mice treated with adenovirus to overexpress GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. E RNAseq quantification heatmap, arranged by calculated hierarchical clustering, demonstrating genes P < 0.05 in db/db AdNAMPT vs. AdGFP mice. n = 4 db/db AdGFP; 4 db/db AdNAMPT mice. Color scale represents Log(FC) ( F ). Most significantly altered KEGG pathways demonstrating log(FC) between db/db AdNAMPT and db/db AdGFP livers. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. Unadjusted P < 0.05 for all pathways shown. G qPCR confirmation of RNAseq data suggesting upregulated liver FGF21 for n = 10 db/db AdGFP; 9 db/db AdNAMPT mice. Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. H and I FGF21 quantification by immunoblot analysis and serum ELISA in db/db AdGFP and db/db AdNAMPT mice. n = 3 db/db AdGFP; 3 db/db AdNAMPT mice for immunoblot data. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice for ELISA data. * P < 0.05, ** P < 0.01 vs. AdGFP. J Glucose tolerance testing and area under the GTT curve in db/db mice treated with adenovirus encoding GFP or NAMPT. n = 9 db/db AdGFP; 9 db/db AdNAMPT biologically independent mice. K Insulin tolerance testing and % area over the ITT curve in db/db mice treated with adenovirus encoding GFP or NAMPT. n = 10 db/db AdGFP; 10 db/db AdNAMPT mice. In ( G – K ), Circles, db/db AdGFP mice, Squares db/db AdNAMPT mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars in ( G , H ) (left), ( I – K ) represent SEM. Statistical tests: ( B , J , K ), repeated measures ANOVA. C , D , G , H , I , J (right panel), ( K ) (right panel) two-tailed T -test. F EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Body weights over time in WT and SIRT1 LKO mice overexpressing empty vector or NAMPT on 12wk Western diet. n = 3 SIRT1 fl/fl Chow; 9 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 5 SIRT1 LKO EV WD. B – D 4 h fasting glucose, insulin, and HOMA-IR (homeostatic model of insulin resistance − fasting glucose (mg/dL) × fasting insulin (ng/mL)/405) in WT and SIRT1 LKO mice overexpressing vector or NAMPT after 12wk chow or Western diet feeding. n = 4 SIRT1 fl/fl Chow; 11 SIRT1 fl/fl EV WD; 4 SIRT1 LKO EV WD; 6 SIRT1 LKO AAV8-NAMPT WD. E FGF21 mRNA and serum peptide, as quantified by qRT-PCR in livers and by serum ELISA in WT and SIRT1 LKO mice overexpressing empty vector or NAMPT after Western diet feeding. Interaction, P < 0.01 and <0.05 for FGF21 mRNA and peptide, respectively. n = 4 SIRT1 fl/fl Chow; 11 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 4 SIRT1 LKO EV WD; 6 SIRT1 LKO AAV8-NAMPT WD ( F ). Indirect calorimetry in WT and SIRT1 LKO mice overexpressing vector or NAMPT after 12wk chow or Western diet feeding. n = 3 SIRT1 fl/fl Chow; 6 SIRT1 fl/fl EV WD; 6 SIRT1 fl/fl AAV8-NAMPT WD; 4 SIRT1 LKO EV WD; 5 SIRT1 LKO AAV8-NAMPT WD. Interactions for light and dark cycle heat: P = ns, and P < 0.01 respectively. *, **, P < 0.05, <0.01 vs. bracketed control by Tukey’s multiple comparisons testing. Error bars in ( A – F ) represent SEM. Circles, SIRT1 fl/fl Chow; Squares, SIRT1 fl/fl EV WD; Upward Triangles SIRT1 fl/fl AAV8-NAMPT WD; Downward Triangles, SIRT1 LKO EV WD; Diamonds SIRT1 LKO AAV8-NAMPT WD. Statistical tests: ( A ), repeated measures ANOVA. B – D one-way ANOVA with Dunnett’s post hoc testing. E , F Two-way ANOVA, Tukey’s post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase

doi: 10.1038/s41467-022-28717-7

Figure Lengend Snippet: A Volcano plots showing differentially expressed genes from (threshold log(FC) = 2, FDR < 0.05) livers from WD-fed SIRT1 fll/fl NAMPT-overexpressing mice and WD-fed SIRT1 fll/fl vector controls (at left). Right, volcano plot showing differentially-expressed genes in livers from WD-fed NAMPT-overexpressing SIRT1 LKO mice vs. NAMPT-overexpressing SIRT1 fll/fl mice. n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. B Gene count of differentially expressed genes in livers from WD-fed SIRT1 fll/fl AAV-NAMPT vs. WD-fed SIRT1 fll/fl vector control mice (Red); differentially expressed genes in WD-fed SIRT1 LKO mice and WD-fed SIRT1 fll/fl mice overexpressing NAMPT (Blue). Common gene count is shown in their intersection (purple). n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. C Left, Unsupervised clustering of differentially expressed genes (Western diet-fed AAV8-NAMPT vs. Western diet-fed AAV8-NAMPT × SIRT1 LKO ). Right, pathway-based gene expression heatmap of the PI3K/AKT signaling pathway, a significantly upregulated GO pathway and CompBio NAMPT-SIRT1-dependent theme. Log 2 (FC) values are Western diet-fed AAV8-NAMPT vs. Western diet-fed AAV8-EV. n = 3 WD-fed SIRT1 fll/fl EV; 3 WD-fed SIRT1 fll/fl NAMPT; 3 WD-fed SIRT1 LKO AAV8-NAMPT. Heatmap color scales represent Log(FC). D Working model of hepatocyte NAMPT signaling and function. NAMPT nicotinamide phosphoribosyltransferase, SIRT1 sirtuin 1, FGF21 fibroblast growth factor 21, GLUT glucose transporter. Statistical tests: ( A , B , D ) EdgeR Exact, Benjamini–Hochberg post hoc correction.

Article Snippet: Sub-cutaneous adipose tissue was dissected from 6 to 8wk-old WT mice and incubated 24 h in regular growth media (DMEM + 4.5 g/L glucose + 10% FCS) with vehicle (0.1% BSA) or with 100 ng/mL recombinant murine FGF21 (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Western Blot, Expressing