infinity total cholesterol reagent  (Thermo Fisher)

Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Western blot analysis of GKRP and GCK in livers from L-GKRPKO mice expressing either human GKRP (hGKRP) or the P446L variant. Samples were collected from mice after an overnight fast followed by 4 hours of refeeding after 13 weeks of HFD feeding. HSP90 was used as a loading control. B-D) Body weight (B), percent fat mass (C), and liver mass (% body weight) of hGKRP- and P446L-expressing mice over 13 weeks of HFD feeding (n = 8 per group). E) Glucose tolerance test on hGKRP- and P446L-expressing mice after 9 weeks on HFD diet (n = 8 per group). F) Plasma insulin levels in mice from (E) at baseline (fasted) and 15 minutes after glucose injection. Samples that measured below the assay’s limit of detection (0.1 ng/mL) were set to 0.1 (marked with a dashed line on the graph). G-L) L-GKRPKO mice expressing hGKRP and hGKRP P446L were fed a HFD for 13 weeks (n = 8 for both groups) G) Hepatic glycogen content after an overnight fast and refeeding for 4 hours. H) Hepatic TAG after overnight fast and refeeding for 4 hours. I) Plasma cholesterol after an overnight fast. J) Plasma cholesterol after an overnight fast and refeeding for 4 hours. K) Plasma TAG from overnight fasted mice. L) Plasma TAG after overnight fast and refeeding for 4 hours. Data are represented as mean ± SEM. Statistical significance was determined using either 2-way ANOVA with either Šidák post hoc test (B, C, and E) or Fisher’s Least Significant Difference (F) or Student’s t test (D, G-L). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: Western Blot, Expressing, Variant Assay, Control, Clinical Proteomics, Injection

Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Volcano plot showing differentially expressed genes identified from RNA-sequencing analysis of livers from control and L-GCKKO mice fed a HCD for 1 week. Samples were obtained from mice that were fasted overnight followed by refeeding for 6 hours. (n = 3/4 control/KO). Red labels: genes upregulated in KO vs. control (FC ≥ 2, adjusted p value < 0.05, total = 44). Green labels: upregulated cholesterol synthesis genes (total = 16). Blue labels: genes downregulated in KO vs. control (FC ≤ −2, adjusted p value < 0.05, total = 15). B) Gene ontology analysis of differentially regulated genes. Labels in each bar represent the number of differentially regulated genes. C) mRNA levels of cholesterogenic genes in livers from control and L-GCKKO mice fed a HCD for 1 week (n = 9/8 control/KO). D-F) Control and L-GCKKO mice were fed a HCD for 1 week. Samples were obtained from mice that were fasted overnight followed by refeeding for 24 hours. (n=10/8 control/KO). D) Total hepatic cholesterol. E) D 2 O-labeled hepatic cholesterol. F) % of labeled cholesterol (of total cholesterol). Data are represented as mean ± SEM. Statistical significance was determined using Student’s t test ( C-F ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: RNA Sequencing, Control, Labeling

Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Western blot analysis of GCK and HKII in livers from control, L-GCKKO, and HKII-overexpressing L-GCKKO mice after 1 week on HCD. Samples were obtained from mice that were fasted overnight followed by refeeding for 24 hours. HSP90 was used as a loading control. B) Glucose tolerance test on control, L-GCKKO, and HKII-overexpressing L-GCKKO mice after 3 days on HCD (n = 9 per group). C) Plasma insulin levels from mice in (B) at baseline (fasting) and 15 minutes after glucose infusion (n = 9 for all groups). Samples that measured below the assay’s limit of detection (0.1 ng/mL) were set to 0.1 (marked with a dashed line on the graph). D-I) Control, L-GCKKO, and HKII-overexpressing L-GCKKO mice fed a HCD for 1 week. Measurements were performed on mice that were fasted overnight, followed by refeeding for 24 hours. D) Hepatic glycogen content. E) Liver mRNA levels of Mlxipl and Pklr . F) Liver mRNA levels of cholesterogenic genes. (D-F control n = 10, L-GckKO n = 9, +HKII n = 10) G) Total hepatic cholesterol. H) D 2 O-labeled hepatic cholesterol. I) % of labeled cholesterol (of total cholesterol). (G-I control n = 7, L-GckKO n = 8, +HKII n = 8). Data are represented as mean ± SEM. Statistical significance was determined using either 2-way ANOVA with Tukey’s post hoc test (B-C) or 1-way ANOVA with Tukey’s post hoc test (D-I). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: Western Blot, Control, Clinical Proteomics, Labeling

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet: (A) Illustration of the CPC experimental system. (B) Plasma cholesterol and triglyceride levels after CPC. Data are presented as mean ± SD. (C) Liver cholesterol and triglyceride levels after CPC normalized on dry tissue weight. Data are presented as mean ± SD. (D) Plasma lipoprotein subclass analysis after CPC. Lipoproteins (vLDL, LDL, IDL, HDL) were separated using fast protein liquid chromatography with in-line analysis of cholesterol (top) and triglyceride (bottom) content in the effluent. Data are presented as mean ± SD. (E) Whole-liver mRNA transcript counting using NanoString. The data are shown as a heatmap with a color scale anchored to the minimum and maximum expression values for each gene. (F) ELISA analysis of IL-1β in plasma after CPC. Data are presented as mean ± SD. (G) Flow-cytometric analysis of Ly6C + and LyC − monocytes subsets in blood, spleen and bone marrow after CPC. Data are presented as mean ± SD. (H) Flow-cytometric analysis of subpopulations of Ly6C + and Ly6C − monocytes in blood. The data are presented in the same manner as in (E). Data were analyzed and p values were obtained by one-way ANOVA with Tukey’s multiple comparisons test or pairwise t test with Holm post hoc correction. n =5 (Ctrl), 6 (Prog), and 8 (Cess) animals in (A)–(F) and n = 5–7 (Ctrl), 6–9 (Prog), and 7–10 (Cess) animals in (G) and (H).

Article Snippet: Total Cholesterol Reagents (Infinity) , Thermo Fisher Scientific , TR13421.

Techniques: Clinical Proteomics, Fast Protein Liquid Chromatography, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet: (A) Relative levels of the metabolites of the TCA cycle in whole aortas from CPC. White/black square on bottom left denotes not detected. (B) SIRM analysis of itaconate isotopologs in CPC aortas from (A). Bottom: scheme illustrating the production of itaconate. Data are presented as mean ± SD. p values were obtained from Student’s t-test. (C) qPCR analysis of the levels of Irg1 transcripts in whole aortas from separate CPC cohorts. N.d., not detected. Data are presented as mean ± SD. p values were obtained from Student’s t-test. (D) Immunofluorescence micrographs depicting positive staining for IRG1 and CD68 in BCA lesions from Prog mice with DAPI counterstain. Scale bars, 100 and 20 μm. (E and F) Histologic assessment with H&E and Movat pentachrome stains and immunofluorescence analysis of IRG1 and CD68 expression in human (F) stable vs. (E) vulnerable thin-cap fibroatheroma (TCFA) plaques from mid-left anterior descending artery (MLAD) with DAPI counterstain. Quantification of co-localization between IRG1 and CD68 is included in (F). Scale bars, 1 mm and 100 μm. (G) BMDMs were treated with free cholesterol for 48 h in the presence or absence of acyl-coenzyme A cholesterol acyltransferase inhibitor (ACAT-i) followed by qPCR mRNA analysis of the expression of Irg1 and Il6 as indicated. Data are presented as mean ± SD. p values were obtained from ANOVA with Tukey post hoc. (H) Immunoblot of IRG1 knockdown in iBMDMs using shRNA. (I) iBMDMs with the IRG1 knockdown (IRG1 KD) or control cells were treated with cholesterol as in (G), and the levels of IkBα and iNOS were determined in the cell lysates using immunoblotting. Staining with Coomassie brilliant blue (CBB) was used as total protein loading control.

Article Snippet: Total Cholesterol Reagents (Infinity) , Thermo Fisher Scientific , TR13421.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Knockdown, shRNA, Control

Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: Total Cholesterol Reagents (Infinity) , Thermo Fisher Scientific , TR13421.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Gene Expression, Software, Microscopy