Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Discovery of Isoginkgetin as a naturally-occurring ACLY inhibitor. (A) Schematic diagram of molecular autodocking. Briefly, we conducted virtual molecular docking based on the structure of the ACLY protein to screen for potential natural small molecules that interact with ACLY from a natural product library comprising 3,200 compounds. (B) The predicted affinity results of A. (C) The inhibitory effect of the top candidates on ACLY enzyme activity was evaluated in vitro using the ADP-Glo assay (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. Compared with compound 0 µM. (D) Chemical structure of ISOGK. (E) The cell viability in primary hepatocytes from C57BL/6J mouse treated with dose-dependent ISOGK detected by CCK-8 assay (n = 4). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (F) The cell viability in HepG2 cell line treated with dose-dependent ISOGK detected by CCK-8 assay (n = 3, technical replicates). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (G) Representative images of Nile Red staining in primary hepatocytes isolated from mice fed a high fat diet for 16 weeks which are treated with vehicle or indicated concentrations ISOGK for 12 h. (H) Quantitative analysis of G (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, Glo Assay, CCK-8 Assay, Staining, Isolation

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin attenuates atherosclerosis in male and female Apoe -/- mice. (A) Scheme of study design. Male and female Apoe -/- mice were induced by high cholesterol diet for 7 weeks. Animals then received ISOGK treatment (20 mg/kg/day) for another 8 weeks. (B) The body weights were shown after ISOGK treatment for 8 weeks (n = 10 or 11). Two-tailed Student's t test. (C) Serum ALT, AST levels of Apoe -/- mice treated with or without ISOGK (n = 10 or 11). Two-tailed Student's t test. (D) Representative images of Oil Red O staining of en face aortas from male and female Apoe -/- mice fed high cholesterol diet. (E) Quantification of en face plaque areas as a percentage of the area in D (n = 10 or 11). Two-tailed Student's t test. (F) The representative images of Oil Red O (upper) staining and H&E (lower) staining of the aortic sinus from male and female Apoe -/- mice fed high cholesterol diet. Arrow indicates necrotic core area. (G) Quantitative analysis of F (n = 6 or 7). Two-tailed Student's t test. The data are means ± SEM, n.s., not significant, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin modulates lipid metabolism in Apoe -/- mouse liver. (A) Scheme of sample processing. (B) Volcano plot showing differential expression genes (adjusted P < 0.05, | log2 (Fold change) | > 1). (C) Heat map displaying the indicated gene expression in liver tissues from Apoe -/- mice with or without ISOGK (n = 4). (D) GO enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (E) KEGG pathway enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (F) GSEA analysis of transcriptomes from Apoe -/- mice liver samples. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Expressing, Gene Expression

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin displays lipid-lowering effects in hyperlipidemia mice and hamsters. (A) Serum levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG from male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (B) The distribution of TC and TG in pooled plasma samples from the indicated female Apoe -/- mice. (C) Study design of the hamster's experiment. (D) Plasma levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG in the indicated hamster's group after treatment ISOGK or vehicle for 4 weeks (n = 8 - 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) The distribution of TC and TG in pooled plasma samples from the indicated hamsters. (F) Representative images of Oil Red O staining in liver sections from the indicated groups. (G) Quantitative analysis of F (n = 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin ameliorates hyperlipidemia and atherosclerosis in Ldlr -/- hamsters. (A) Scheme of study design. Briefly, 8-week-old male Ldlr -/- hamsters were induced by high cholesterol diet for 2 weeks and then administrated ISOGK (2 mg/kg/day and 5 mg/kg/day) treatment for another 6 weeks. (B) Hepatic levels of TC, TG in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (C) Serum TC, TG, HDL-C and non-HDL-C levels in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 6 weeks (n = 7). One-way ANOVA followed by Bonferroni's post hoc test. (D) Representative images of Oil Red O staining of en face aortas in indicated Ldlr -/- hamsters group. (E) Quantitative analysis of D (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Representative images of Oil Red O staining in Ldlr -/- hamsters' aortic sinus and representative images of Nile Red staining in Ldlr -/- hamsters' liver tissues. (G-H) Quantitative analysis of F (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin inhibits ACLY activity in vitro and in vivo. (A) Primary hepatocytes from C57BL/6J mice were cultured with vehicle or 5 µM ISOGK for 4 h in the presence of [1,2- 14 C]-acetate. The hepatic lipogenesis was calculated (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (B) Dose-dependent inhibition of purified, recombinant ACLY after incubation with ISOGK, the activity at control was defined as 100%. (C) ACLY enzyme activities were measured from the liver extracts of male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (D). ACLY enzyme activities were measured from the liver extracts of hamster after treatment ISOGK or vehicle for 4 weeks (n = 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) ACLY enzyme activities were measured from the liver extracts of Ldlr -/- hamster after treatment with or without ISOGK for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Molecular docking has predicted the binding sites of ISOGK with ACLY. (G) The interaction of ACLY with ISOGK was measured by surface plasmon resonance. (H) CETSA analyzed the thermal stabilization of ACLY with isoginkgetin in AML12 cell lysates. (I) Quantitative analysis of H (n = 3). Two-way ANOVA with Dunnett's T3 post hoc analysis. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, In Vivo, Cell Culture, Inhibition, Purification, Recombinant, Incubation, Control, Two Tailed Test, Binding Assay, SPR Assay

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: The anti-atherosclerotic and lipid-lowering effects of isoginkgetin in Apoe -/- mice were ACLY-dependent. (A) Scheme of study design. Briefly, male Apoe -/- mice were induced by high cholesterol diet for 7 weeks and administrated GalNAc-siAcly (3 mg/kg/month). Animals then received ISOGK (20 mg/kg/day) treatment for another 8 weeks. (B) Immunoblotting analysis and quantification of ACLY protein in liver and kidney tissues from indicated groups (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (C) Representative images of Oil Red O staining of en face aortas from male Apoe -/- mice fed high cholesterol diet. (D) Representative images of ORO staining of the aortic sinus from male Apoe -/- mice fed high cholesterol diet. (E) Representative images of H&E staining of the aortic sinus from male Apoe -/- mice fed High cholesterol diet. (F) Quantitative analysis of C-E (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. (G) Plasma levels of TC, TG, LDL-C and HDL-C in the indicated groups (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Western Blot, Staining

Journal: Molecular cell

Article Title: Alternative splicing is a developmental switch for hTERT expression

doi: 10.1016/j.molcel.2021.03.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal Anti-SON Novus Biologicals NBP1-88706 Mouse monoclonal GAPDH (0411) Santa Cruz Biotech. sc-47724 Mouse monoclonal SSEA-4 (MC-813–70) R&D Systems MAB1435 Mouse monoclonal COL1A1 (3G3) Santa Cruz Biotech. sc-293182 Bacterial and virus strains Cytotune iPS Sendai Reprogramming kit ThermoFisher A16517 One Shot Stbl3 Chemically Competent E. coli ThermoFisher C737303 Biological samples Patient derived PBMCs Dr. Savage, NIH NCT-00027274 Chemicals, peptides, and recombinant proteins Y-27632 2HCl SelleckChem S1049 Isoginkgetin Tocris 6483 Critical commercial assays Dual-Luciferase® Reporter Assay System Promega E1910 Nano-Glo® Dual-Luciferase® Reporter Assay System Promega N1610 DNA Prep Kit, Tagmentation Illumina 20018704 Deposited data Raw and analyzed sequencing data This paper GEO accession: GSE168193 Human reference genome NCBI build 37, GRCh37 Genome Reference Consortium https://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/ Raw data at Mendeley This paper https://dx.doi.org/10.17632/jfmsvxnpbg.1 Experimental models: cell lines Human: BJ Fibroblast ATCC CRL-2522 Human: ARPE-19 ATCC CRL-2302 Human: HeLa ATCC CCL-2 Human: TREx-HeLa Invitrogen R71407 Human: H7 ESC Dr. Lei Bu WA07 Oligonucleotides See Table S1 This paper N/A Recombinant DNA pRF-HCV Maria Barna n/a pNL1.1 Promega N1001 pcDNA5/FRT/TO ThermoFisher V652020 Software and algorithms 4C-ker Raviram et al., 2016 https://github.com/rr1859/R.4Cker Oligostan Tsanov et al., 2016 https://bitbucket.org/muellerflorian/fish_quant ImageJ Schneider et al., 2012 https://imagej.nih.gov/ij/ Bowtie2 Langmead and Salzberg, 2012 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml Samtools Li et al., 2009 http://samtools.sourceforge.net/ Open in a separate window KEY RESOURCES TABLE Enhancers have a minor role in regulating telomerase during development Alternative splicing of exon 2 is a major determinant of hTERT levels Exon 2 skipping triggers hTERT mRNA decay in somatic cells SON regulates hTERT splicing, and its mutation manifests in telomerase insufficiency

Techniques: Virus, Derivative Assay, Recombinant, Reporter Assay, Sequencing, Software

Journal: Nucleic Acids Research

Article Title: Tracing the molecular basis of transcriptional dynamics in noisy data by using an experiment-based mathematical model

doi: 10.1093/nar/gku1272

Figure Lengend Snippet: Re-fitting the model can indicate the perturbation in the system. Human HepG2 cells were pre-incubated with 100 μM isoginkgetin for 16 h and subsequently treated with PPARδ ligand for the indicated time periods. qPCR was performed in order to measure pre-mRNA ( A ) and mature mRNA levels ( B ) and copy numbers were calculated. Small filled circles represent all data points from at least four biological experiments corrected for outlier and empty circles indicate their mean. All the individual data points are reported in Supplementary Tables S9. The mathematical model (adjusted for apparent fold induction) was fitted to the data by changing a single constant at a time and the quality of the fit was compared for pre-mRNA (grey) and mature mRNA (green) time courses ( C ). The best model obtained by lowering the splicing rate constant was further tested for fit improvement by changing the remaining constants ( D ). The best fit given by adjusting both splicing and degradation constants is also shown for the pre-mRNA (A) and mature mRNA (B) time courses.

Article Snippet: The splicing inhibitor isoginkgetin (kindly provided by the Dr Willmar, Schwabe GmbH & Co. KG, Karlsruhe, Germany) was added after 24 h to a final concentration of 50 μM.

Techniques: Incubation