Fig 1: Continuous collection of high-quality CSF from naturally-behaving mice. a Quantitative assessment of CSF blood contamination using a highly sensitive spectrometer method. Hemoglobin contamination levels (absorbance at 417 nm) from blood-spiked CSF (intentionally spiked with 1%, 0.1%, or 0.01% vol/vol of blood, black), mouse CSF collected by the conventional single collection technique (blue), and cCSF at various postoperative times (red). Insets are representative images of spiked CSF and cCSF. b Direct blood count under the phase contrast microscope for sensitive detection of CSF blood contamination. (b, left) Representative images of red blood cell (RBC) contamination in the blood-spiked CSF, single collection CSF, and cCSF. The arrows indicate RBCs. (b, right) The RBC count in the CSF samples (n = 3–4). c ELISA measurement of plasma abundant protein ApoB in the single collection CSF, cCSF, and plasma. Plasma samples were diluted to 1:1,000, 1:10,000, 1:100,000; CSF samples were diluted to 1:100 for the measurements. d Normal cCSF electrolyte concentrations during long-term continuous collection. Sodium, potassium, and chloride levels measured in the single-collection CSF and cCSF at various postoperative times (n = 4–6, one-way ANOVA). e, f) Quantifications of albumin (e) and ApoE protein (f) in the single collection CSF and cCSF (n = 4–10). g Quantification of human total tau levels in the single collection CSF and cCSF. Seven-month-old tau-transgenic PS19 mice and wild-type littermates were used for the experiment (n = 3–7). h Mice’s normal locomotor activities and circadian rhythms during long-term cCSF collection. The locomotor activities were measured every 3 h before (baseline, black) and during cCSF (red) under the normal 12:12 h light-dark cycle. i Locomotor activity dark/light ratio during baseline and cCSF is shown (n = 3, Student’s t-test). CSF cerebrospinal fluid, AOM atlanto-occipital membrane, i.d. inner diameter, cCSF CSF obtained by the continuous collection method, n.d. not detected, n.s. not significant
Fig 2: DSHT therapy promotes cholesterol metabolism in serum and enhances the expression of ABCA1, ABCG1, and LDLR in the aorta. Serum (A) ApoA1 and (B) ApoB levels, and (C) their ratio. (D) Relative mRNA expression levels of cholesterol metabolism-related ABCA1, ABCG1, and LDLR genes. Data are presented as mean ± standard error of mean of the samples from mice in the vehicle, AT, DSHT, and DSHT + AT groups (n = 5 for all). * p < 0.05; ** p < 0.01; *** p < 0.001, compared with the vehicle group (Mann–Whitney U test).
Fig 3: Hepatic lipoprotein and APOB secretion defect in Surf4fl/fl Alb-Cre+ mice.(A) Steady-state serum APOB levels in control and Surf4fl/fl Alb-Cre+ mice at 2 months of age (n=4 per genotype). (B) Representative immunoblot for APOB and HSP90 in liver lysates with and without endoglycosidase H (endo H) treatment. Proteins in the pre-Golgi compartments are expected to be sensitive to endo H cleavage, resulting in an electrophoretic shift on an immunoblot. Blue arrowhead indicates the endo H resistant band whereas the red arrowhead indicates the endo H sensitive band. Molecular weight markers notated are in kDa. Accumulation of endo H sensitive APOB in the absence of SURF4 suggests accumulation in the ER (Figure 3—source data 1). (C) Quantification of APOB abundance in control and Surf4fl/fl Alb-Cre+ liver lysates, without endo H treatment (n=4 per genotype). For panel A and C, crossbars represent the mean, with statistical significance determined by two-sided Student’s t-test. (D–G) Surf4fl/fl Alb-Cre+ and littermate control mice were injected with a lipoprotein lipase inhibitor to block triglyceride hydrolysis. Blood was sampled prior to and following injection over 24 hr and assayed for (D) glucose, (E) cholesterol, (F) triglycerides, and (G) APOB levels. Data are presented as mean ± SEM for each time point (n=5 per genotype). Asterisks denote p<0.05 obtained from two-sided Student’s t-test with Benjamini-Hochberg adjustment for multiple hypothesis testing, n.s., not significant. Figure 3—source data 1.Uncropped and unedited blots shown in Figure 3.
Fig 4: Body composition, food intake and systemic metabolic parameters during NAFLD development. Groups of Ldlr-/-. Leiden mice fed a chow or a high-fat (HFD) diet were euthanized over time up to 38 weeks. (A) body weight and caloric food intake, (B) Fat mass and lean mass determined with echoMRI, (C) visceral fat mass is a composite of the visceral epididymal and mesenteric fat mass. In 5-h fasted plasma (D) cholesterol and triglyceride concentrations were determined as well as (E) insulin and glucose in whole blood. Insulin and glucose concentrations were used to calculate the (F) HOMA-IR. Lipoprotein profiles were analyzed in fasting plasma pools (n = 15/grp) and fractionated with FPLC, in the respective fractions (G) cholesterol and (H) triglyceride concentrations were determined at t = 20 and plotted as profiles. (I) plasma apoB concentrations. Circulating liver integrity markers (J) AST and ALT were determined in plasma pools (n = 8/grp). Liver damage markers including (K) CK-18M30 and TIMP1 were analyzed in 5-h fasted plasma. The x-axis indicates the time in weeks and data represent mean ± SEM with * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD.
Fig 5: A small molecule screen using human iPSC-derived hepatocytes.a Schematic overview of the procedure used to identify compounds that reduce apoB produced by human iPSC-hepatocytes. b Graph showing post-drug: pre-drug ratios (? [apoB]) in the medium of apoB in compound-treated compared to DMSO-treated iPSC-hepatocytes (% DMSO). The blue dots represent each individual compound from the SC3 library 10 K representative set. c Graph showing the z score of each individual compound (blue dots) on the post-drug: pre-drug apoB ratio. Red dot is DL-1. Red line indicates the z-score at a value of –2.75 (z = –2.75).
Supplier Page from Abcam for Mouse Apo B ELISA Kit