Fig 2: Specific enzymatic activity of (A) aldolase (ALDOA), (B) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (C) phosphoglycerate kinase (PGK1), (D) enolase (ENO) and (E) pyruvate kinase M1/2 (PKM) in cell-free extract (CFE) and their corresponding sEV fractions. The bar graph represents mean values, while error bars indicate the standard deviation (SD) of at least five replicates, and p-values obtained from a Student’s t-test are indicated on the top.

Fig 3: Genetic loss of Aldoc reduces plasma cholesterol in mice. A: Relative liver mRNA expression of Hmgcr, Aldoc, Aldoa, and Aldob in mice fed a control diet or diet containing 0.2% cholesterol. B: Relative liver mRNA expression of Hmgcs1, Aldoc, Aldoa, and Aldob in the ad-lib fed mice or 20-h fasted mice. (C) Plasma and (D) liver concentrations of cholesterol and triglycerides in male 16-week-old Aldoc−/− and Aldoc+/+ littermate control mice fasted for four hours. E: Analysis of cholesterol-containing lipoproteins by FPLC fractionation of plasma from male 16-week-old Aldoc−/− and Aldoc+/+ littermate control mice fasted for four hours. (F) Plasma and (G) liver concentrations of cholesterol and triglycerides in female 16-week-old Aldoc−/− and Aldoc+/+ littermate control mice fasted for four hours. H: Analysis of cholesterol-containing lipoproteins by FPLC fractionation of plasma from female 16-week-old Aldoc−/− and Aldoc+/+ littermate control mice fasted for four hours. Data presented as mean ± SD. Statistical differences were determined with an unpaired two-tailed t test denoted by ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Aldoc, aldolase C; FPLC, fast-protein liquid chromatography; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Hmgcs1, hydroxymethylglutaryl-CoA synthase 1.

Fig 4: Validation of Aldoc as a cholesterol-related gene. A: Relative mRNA expression of Hmgcs1 and Aldoc in mouse AML12 hepatocyte cells incubated with 10% FBS and 3 different concentrations of human LDL-C. B: Analysis of cellular cholesterol concentration in mouse AML12 hepatocyte cells after being transfected with either control (Scrambled) or Aldoc siRNA and incubated with 0% FBS, 5% LPDS for 16 h, or 4 h with 0.5% MBCD/5% LPDS followed by 4 h with 5% LPDS. Analysis of glucose or acetate converted into cholesterol in mouse AML12 hepatocyte cells transfected with control (Scrambled) or Aldoc-directed siRNA and treated for 4 h with 0.5% MBCD/5% LPDS and 4 h with 5% LPDS with (C) 1 μCi of glucose D-[6–14C] or (D) 1 μCi of acetic acid [U-14C]. E: Relative mRNA expression of Hmgcs1 in mouse AML12 hepatocytes represented as percent change from control after transfection with control (Scrambled), Aldoc, Aldoa, or Aldob-directed siRNAs after incubation with 0.5% MBCD/5% LPDS for 4 h. F: Analysis of glucose converted into cholesterol in mouse AML12 hepatocyte cells transfected with control (Scrambled), Aldoc, Aldoa, or Aldob-directed siRNAs and treated for 4 h with 0.5% MBCD/5% LPDS and 4 h with 5% LPDS with μCi of glucose D-[6–14C]. G: Relative abundance of [U-13C] labeled polar metabolites in mouse AML12 cells transfected with control (Scrambled) or Aldoc-directed siRNA and treated with 0.5% MBCD/5% LPDS for four hours followed by treatment with 5% LPDS and isotopically labeled 13C glucose for four hours. H: Aldolase activity in mouse AML12 hepatocyte cells transfected with control (Scrambled) or Aldoc-directed siRNA and treated with either 10% FBS or 0.5% MBCD/5% LPDS for four hours. I: Total intracellular 14C quantification in mouse AML12 hepatocyte cells transfected with control (Scrambled) or Aldoc-directed siRNA and treated with 0.5% MBCD/5% LPDS for four hours followed by treatment with 5% LPDS and 1 μCi of glucose D-[6–14C] for four hours. Data presented as mean ± SD. Statistical differences in A, B, and F were determined by one-way ANOVA with Tukey’s post hoc test. Statistical differences in C to E were determined with an unpaired two-tailed t test, both denoted by ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Aldoc, aldolase C; AML12, alpha mouse liver 12; Hmgcs1, hydroxymethylglutaryl-CoA synthase 1; LDL-C, low density lipoprotein cholesterol; LPDS, lipoprotein deficient serum, MBCD, methyl-beta cyclodextrin.

Fig 5: Specific enzymatic activity of (A) Glutathione S-transferase (GST), (B) Aldolase (ALDOA), and (C) Sorbitol dehydrogenase (SORD) from frog liver. The bar graph represents the mean value of the biological replicates, whereas error bars indicate the standard deviation of the replicates, and p-values obtained from a Student’s t-test are indicated on the top. Data were considered statistically significant if p < 0.05.