Journal: Advanced Science

Article Title: Depletion of Hepatic SREBP2 Protects Against Hypercholesterolemia and Atherosclerosis through the ANGPTL3‐LPL Axis

doi: 10.1002/advs.202412677

Figure Lengend Snippet: Depletion of hepatic SREBP2 promotes Lipoprotein lipase activity in the absence of LDLR. A,B) Plasma lipoprotein lipase contents (A) and lipoprotein lipase activity (B) measured in heparinized C57BL/6J or Ldlr −/− male mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under chow diet feeding ( n = 8 mice per group, by two‐way ANOVA). C,D) Plasma lipoprotein lipase contents (C) and lipoprotein lipase activity (D) measured in heparinized Ldlr −/− male mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under 5‐week chow diet feeding followed by 3‐week western diet feeding ( n = 6–8 mice per group, by student's t test). E) Relative expression of the genes involved in de novo lipogenesis, cholesterol synthesis and LPL regulation measured by RNA‐seq using different CRISPR/Cas9‐mediated genetically modified AML12 mouse hepatocyte cell line ( n = 3 replicates for each cell line). F,G) qPCR expression analysis of genes modulating LPL activity in the liver of C57BL/6J (F) and Ldlr −/− (G) mice injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses under 5‐week chow followed by 5‐week western diet feeding ( n = 5–6 mice per group, by student's t test). H) qPCR expression analysis of ANGPTL3 , ANGPTL4 , and ANGPTL8 in SREBF2 knockout and control Huh7 human hepatoma cells ( n = 3 replicates for each cell line, representative of 3 independent experiments, by student's t test). I) qPCR expression analysis of ANGPTL3 , ANGPTL4 , and ANGPTL8 in SREBF2 knockout or control Huh7 human hepatoma cells lacking LDLR ( n = 3 replicates for each cell line, representative of 3 independent experiments, by student's t test). J–M) Western blot (J and L) and quantification (K and M) of ANGPTL3 (FL stands for full length) and ANGPTL8 in heparinized plasma of 4h‐fasted male C57BL6J mice (J and K, n = 7 mice per group, by t student's t test) and mice lacking LDLR (L and M, n = 6–8 mice per group, by student's t test) injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses fed chow. N,O) Western Blot of cellular (N) and secreted (O) ANGPTL3 protein in SREBF2 knockout and control LDLR +/+ and LDLR −/− Huh7 human hepatoma cells (representative of 3 independent experiments). P,Q) Western blot and relative quantification of ANGPTL3 in heparinized plasma of 4 h fasted male C57BL/6J mice and mice lacking LDLR injected with AAV8‐scramble control or AAV8‐ Srebf2 ‐shRNA viruses fed chow (3 representative mice of each group consisting of 6–8 mice, by one‐way ANOVA). * p < 0.05, ** p < 0.01, and *** p < 0.001; Error bars indicate mean ± SD.

Article Snippet: The AML12 (alpha mouse liver 12) cell line was established from hepatocytes obtained from a male mouse (CD1 strain, line MT42) transgenic for human TGF alpha (ATCC, ATCC CRL‐2254).

Techniques: Activity Assay, Clinical Proteomics, Injection, Control, shRNA, Western Blot, Expressing, RNA Sequencing, CRISPR, Genetically Modified, Knock-Out, Quantitative Proteomics

Journal: Cell reports

Article Title: Non-cell-autonomous regulation of mTORC2 by Hedgehog signaling maintains lipid homeostasis

doi: 10.1016/j.celrep.2024.115191

Figure Lengend Snippet: (A) A western blot analysis of lysates from AML12 mouse hepatocytes challenged with Smoothened agonist (SAG) for the indicated times. Insulin treatment is a positive control for AKT phosphorylation. (B–G) Quantification of western blot images for the indicated phosphoproteins relative to their total protein levels (mean ± SEM; * p < 0.05; ** p < 0.01; n.s., not significant; t test). The GLI1 protein is normalized to actin levels. Either the 10- or 15-min time point was used for quantification (see ). (H) A western blot analysis of lysates from cells transfected with either mock or Rictor siRNAs and challenged with vehicle, 0.5 μM SAG (15 min), or 100 nM insulin (30 min). (I) DEGs in the rict-1(mg360) mutant compared to the grd triple mutant (hypergeometric p value reported). (J) A scatterplot showing the differential expression values for the rict-1(mg360) mutant plotted against the grd mutant ( rict-1 -specific DEGs in red, grd -specifc DEGs in blue, and rict-1/grd co-regulated genes in black). R 2 values are reported for linear regression analyses on the union and intersection of the rict-1 and grd DEG datasets. (K and L) (K) Representative images (scale bar, 50 mm; white arrowheads indicate nuclei) and (L) quantification of DAF-16::mKate2 and GFP::PQM-1 nuclear fluorescence in day 1 adult WT and grd mutant animals reared at 20°C (mean ± SD; **** p < 0.0001; t test). (M) The average DAF-16 (left) or PQM-1 (right) enrichment (ChIP-seq signal) at the transcriptional start site (TSS) at all genes (red) or at genes differentially expressed in the grd mutant (black). See also .

Article Snippet: The alpha mouse liver 12 (AML12) hepatocyte cell line was isolated from a 3-month-old male mouse liver (CD1 strain, line MT42) transgenic for human TGFα (CRL-2254, ATCC).

Techniques: Western Blot, Positive Control, Phospho-proteomics, Transfection, Mutagenesis, Quantitative Proteomics, Fluorescence, ChIP-sequencing

Journal: Cell reports

Article Title: Non-cell-autonomous regulation of mTORC2 by Hedgehog signaling maintains lipid homeostasis

doi: 10.1016/j.celrep.2024.115191

Figure Lengend Snippet: (A) The rict-1/grd co-regulated genes are enriched for pmk-1 -dependent genes (hypergeometric p value reported ). (B) Quantification of lipid levels using Nile Red staining (day 1 adults reared at 20°C; mean ± SD; ** p < 0.01; *** p < 0.001; **** p < 0.0001; one-way ANOVA). (C) A heatmap showing differential expression values (log2 fold change relative to WT) of the 1,632 grd -dependent genes ( R 2 values are shown for the indicated comparisons). (D–G) Western blot and quantification of phospho-p38/PMK-1 levels for (D and E) C. elegans lysates prepared from WT, rict-1(mg360) , grd , or nsy-1(ums8) mutants ( ums8 is a gain-of-function allele; mean ± SEM; * p < 0.05; n.s., not significant; one-way ANOVA) and (F and G) AML12 hepatocytes treated with SAG for increasing amounts of time (10- or 15-min time point quantified; mean ± SEM; ** p < 0.01; t test). (H) A model of how Hh governs intestinal metabolism through the dual regulation of mTORC2 and p38 signaling in C. elegans . See also .

Article Snippet: The alpha mouse liver 12 (AML12) hepatocyte cell line was isolated from a 3-month-old male mouse liver (CD1 strain, line MT42) transgenic for human TGFα (CRL-2254, ATCC).

Techniques: Staining, Quantitative Proteomics, Western Blot

Journal: Cell reports

Article Title: Non-cell-autonomous regulation of mTORC2 by Hedgehog signaling maintains lipid homeostasis

doi: 10.1016/j.celrep.2024.115191

Figure Lengend Snippet: key resources table

Article Snippet: The alpha mouse liver 12 (AML12) hepatocyte cell line was isolated from a 3-month-old male mouse liver (CD1 strain, line MT42) transgenic for human TGFα (CRL-2254, ATCC).

Techniques: Virus, Recombinant, SYBR Green Assay, Transfection, DC Protein Assay, Mutagenesis, CRISPR, Plasmid Preparation, Software