Journal: Medicina

Article Title: Retrospective Analysis of a Quince, Olive Leaf, and Amaranth Nutraceutical in Patients with Metabolic Syndrome

doi: 10.3390/medicina61091638

Figure Lengend Snippet: Preclinical evaluation of QUINOLAM on metabolic and cellular endpoints in HepG2 cells. ( A ) Cell viability assessed by MTT assay after 24 h exposure to increasing concentrations of QUINOLAM (0.003 to 1.6 mg/mL). No cytotoxic effects were observed up to 1.6 mg/mL. SDS (1 mg/mL) was used as a positive control. ( B ) Glucose uptake measured using the 2-NBDG fluorescent analog following 24 h treatment with QUINOLAM at 0.5, 1.0, and 2.5 mg/mL. A dose-dependent increase in glucose uptake was observed, with significant enhancement at 2.5 mg/mL ( p < 0.01 vs. control). ( C ) LDL receptor (LDL-R) protein expression determined by ELISA after 24 h exposure to QUINOLAM. Treatments with 1.0 and 2.5 mg/mL significantly upregulated LDL-R levels compared to untreated controls ( p < 0.05). ( D ) Antioxidant activity of QUINOLAM evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. QUINOLAM displayed strong antioxidant potential in a dose-dependent manner, with significant increases in TEAC values at 0.1 and 0.2 mg/mL (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control). All data are expressed as mean ± standard deviation from three independent experiments.

Article Snippet: All in vitro experiments were conducted using the HepG2 human hepatic cell line (ATCC, Manassas, VA, USA).

Techniques: MTT Assay, Positive Control, Control, Expressing, Enzyme-linked Immunosorbent Assay, Antioxidant Activity Assay, Standard Deviation

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 1 Synthetic lethal CRISPR screening to identify potential cholesterol regulators in HepG2 cells. A Immunoblot analysis of HMGCR in HepG2 cells undergoing CRISPR-mediated gene knockout with two independent sgRNAs (numbered as _1 and _2). Vector without specific sgRNA insert serves as a control. GAPDH serves as a loading control. B Immunoblot analysis of LDLR in HepG2 cells that have undergone CRISPR-mediated gene knockout with two independent sgRNAs. C The cell growth analysis of HepG2 cells after introducing indicated sgRNAs via lentiviral infection for 7 days. Cells were counted with a hemacytometer. Mean ± SD with n = 3. Ordinary one-way ANOVA with Tukey’s test, **p < 0.01, ***p < 0.001. D The relative cell viability was determined by CCK-8 assay for HepG2 cells expressing indicated sgRNAs and treated with indicated doses of lovastatin. Mean ± SD with n = 6. Ordinary one-way ANOVA with Dunnett’s test, *p < 0.05, **p < 0.01, ***p < 0.001. E The workflow of genome-scale synthetic lethal CRISPR screens (Screen 1) to identify negative GIs with HMGCR using its inhibitor lovastatin in HepG2 cells. F The scatter plot showing the β score of each gene and the correlation of both CRISPR screens (vehicle and lovastatin) in HepG2 cells. The genes in blue box are preferential targets as the synthetic lethal or negative GI hits. G The rank-ordered list of each gene in the CRISPR screens according to the strength of synthetic lethality measured by differential β scores between lovastatin and vehicle conditions. The top interesting gene hits are highlighted. H The top selected functional terms enriched among synthetic lethal hits of the CRISPR screens (Screen 1) as determined by the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: CRISPR, Western Blot, Gene Knockout, Plasmid Preparation, Control, Infection, CCK-8 Assay, Expressing, Functional Assay

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 3 Identification of potential cholesterol regulators at the transcriptional level. A Venn diagram showing the overlap of up- or down-regulated DEGs of HepG2 cells upon LDLR KO, HMGCR, or double KO (DKO) determined by RNA-seq analysis. B Heatmap showing the DEGs across different samples of HepG2 cells. C Volcano plot showing the DEGs in DKO cells compared to control HepG2 cells with several typical genes highlighted. D The top five enriched functional terms for either up-regulated or down-regulated DEGs in DKO cells compared to control HepG2 cells by GO and KEGG analysis. E Venn diagram showing the overlap of DEGs between sterol deprivation condition (sterol depleted vs. normal) and SREBF2 KO under sterol deprivation (SREBF2 KO vs. vector control) in HepG2 cells. F Heatmap showing the DEGs across indicated samples of HepG2 cells. G Volcano plot showing the DEGs between sterol deprivation and normal conditions of HepG2 cells with several representative genes highlighted. H Volcano plot showing the DEGs in SREBF2 KO cells compared to vector control HepG2 cells in sterol deprivation condition with several representative genes highlighted. I Venn diagram showing the overlap of DEGs for indicated comparison groups. J Heatmap showing the expression change of representative DEGs that were shared between at least three comparison groups of I

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: RNA Sequencing, Control, Functional Assay, Plasmid Preparation, Comparison, Expressing

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 4 Integrative multi-omics analysis to pinpoint key cholesterol regulators. A Heatmap showing the differentially expressed proteins in LDLR/HMGCR DKO cells compared to Vector control HepG2 cells determined by mass spectrometry-based proteomics profiling. The number of up- or down-regulated proteins is indicated along the heatmap. B Venn diagram showing the overlap of either up-regulated or down-regulated genes in LDLR/HMGCR DKO cells compared to Vector control HepG2 cells between RNA-seq and proteomics analysis. C Volcano plot showing the differentially expressed proteins in LDLR/HMGCR DKO cells compared to control HepG2 cells with several typical proteins highlighted. D The top ten enriched functional terms for up-regulated proteins in LDLR/HMGCR DKO cells compared to control HepG2 cells by GO and KEGG analysis. E The analytic scheme of human genes associated with lipid disorders or cardiovascular diseases. HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low-density lipoprotein; TC: total cholesterol; TG: triglycerides; CAD: coronary artery disease. F Venn diagram showing the overlap of potential cholesterol or lipid regulators between different angles including synthetic lethal CRISPR screens (Screen 1–3), DEGs in RNA-seq analysis (DKO vs. vector in HepG2 and HeLa cells, sterol depleted vs normal, and SREBF2 KO vs vector), differentially expressed proteins in proteomics analysis (DKO vs. vector in HepG2 cells), and the compiled list of human genes with variants related to lipid disorders or related diseases. The genes of indicated intersections are highlighted

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Biomarker Discovery, Plasmid Preparation, Control, Mass Spectrometry, RNA Sequencing, Functional Assay, CRISPR

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 5 Functions of GGT7 during cholesterol and lipid homeostasis. A RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon LDLR KO, HMGCR KO, or LDLR/HMGCR DKO. Mean ± SD with n = 3. Ordinary one-way ANOVA with Dunnett’s test, compared to Vector control, **p < 0.01, ***p < 0.001. B RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon SREBF2 KO under sterol deprivation condition. Mean ± SD with n = 3. Unpaired two-sided t test, compared to vector control, **p < 0.01, ***p < 0.001. C Immunoblot analysis of GGT7 in HepG2 cells undergoing CRISPR-mediated gene knockout with two independent sgRNAs. GAPDH serves as a loading control. D Decreased total cholesterol levels of HepG2 cells upon GGT7 KO using two independent sgRNAs. Mean ± SD with n = 3. Unpaired two-sided t test, compared to vector control, *p < 0.05. E Decreased sterol and other lipids upon GGT7 KO in HepG2 cells determined by untargeted metabolomic profiling. n = 6 biological replicates for each group

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: RNA Expression, Quantitative RT-PCR, Plasmid Preparation, Control, Western Blot, CRISPR, Gene Knockout

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 6 Mechanistic insights of GGT7 in regulating cholesterol metabolism. A RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon GGT7 KO. Mean ± SD with n = 3. Unpaired two-sided t test, compared to Vector control, *p < 0.05, **p < 0.01, ***p < 0.001. B RNA expression analysis of indicated genes by RT-qPCR in HepG2 cells upon GGT7 KO under normal or sterol deprivation conditions. Mean ± SD with n = 3. Unpaired two-sided t test, *p < 0.05, **p < 0.01, ***p < 0.001. C Volcano plot showing the DEGs in GGT7 KO cells compared to vector control HepG2 cells with several typical genes highlighted. The number of up- or down-regulated DEGs is indicated. D The top selected functional terms enriched for down-regulated DEGs in GGT7 KO cells compared to control HepG2 cells by GO and KEGG analysis. E Coomassie blue staining of SDS-PAGE gel with FLAG bead immunoprecipitated materials from vector control- or FLAG-GGT7-expressing HepG2 cells. The band position corresponding to GGT7 or MYH10 is indicated with an asterisk. F The top ten list of GGT7-interacting protein partners identified by mass spectrometry. G Immunoblot analysis of total cell lysis and immunoprecipitants (using IgG control, GGT7, or MYH10 antibody) for indicated proteins derived from HepG2 cells. H Immunoblot analysis of MYH10 in HepG2 cells that have undergone CRISPR-mediated gene knockout. GAPDH serves as a loading control. I Decreased total cholesterol levels of HepG2 cells upon MYH10 knockout. Mean ± SD with n = 3. Unpaired two-sided t test, compared to AAVS1 KO, *p < 0.05. Decreased Dil-LDL uptake by HepG2 cells upon J GGT7 or K MYH10 knockout. Mean ± SD with n = 3. Unpaired two-sided t test, compared to AAVS1 KO, **p < 0.01, ***p < 0.001

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: RNA Expression, Quantitative RT-PCR, Plasmid Preparation, Control, Functional Assay, Staining, SDS Page, Immunoprecipitation, Expressing, Mass Spectrometry, Western Blot, Lysis, Derivative Assay, CRISPR, Gene Knockout, Knock-Out

Journal: Genome biology

Article Title: Systematic interrogation of functional genes underlying cholesterol and lipid homeostasis.

doi: 10.1186/s13059-025-03531-8

Figure Lengend Snippet: Fig. 7 Impaired cholesterol and lipid homeostasis in Ggt7 knockout mice. A The construction strategy of whole-body Ggt7 KO mice using two sgRNAs targeting the flanks of exon 2 and exon 3 of Ggt7 in the mouse genome. B Immunoblot analysis of Ggt7 in different types of tissues derived from Ggt7+/+ and Ggt7−/− mice. β-actin serves as a loading control. C Body weight measurements for Ggt7+/+ and Ggt7−/− mice under normal diet (ND) (n = 12 and 10, respectively) or high-fat high cholesterol (HFHC) diet (n = 7 and 8, respectively). Unpaired two-sided t test, *p < 0.05, ns means not significant. D The serum total cholesterol levels (n = 12, 10, 7 and 8 for each group), E serum HDL-cholesterol (HDL-C) levels (n = 10, 10, 7 and 8), F serum LDL-cholesterol (LDL-C) levels (n = 11, 10, 7 and 8) and G serum triglycerides (TG) levels (n = 12, 10, 7 and 8) in Ggt7+/+ and Ggt7−/− mice fed with ND or HFHC diet. Unpaired two-sided t test, *p < 0.05, **p < 0.01, ns means not significant. H The total cholesterol (TC) levels (n = 12, 10, 7 and 7) or I TG levels (n = 12, 10, 7 and 8) in the livers of Ggt7+/+ or Ggt7−/− mice fed with ND or HFHC diet. Unpaired two-sided t test, ns means not significant. J The TC levels (n = 12, 10, 7, and 7) or K TG levels (n = 12, 10, 7, and 7) in the brain tissues of Ggt7+/+ or Ggt7−/− mice fed with ND or HFHC diet. Unpaired two-sided t test, **p < 0.01, ns means not significant. L Volcano plot showing the DEGs upon Ggt7 knockout in the mouse liver or brain tissues. The number of up- or down-regulated DEGs is indicated. M Venn diagram showing the overlap of DEGs between Ggt7 KO in mouse liver, Ggt7 KO in mouse brain, and GGT7 KO in HepG2 cells as determined by RNA-seq analysis. N The top five enriched functional terms for either up-regulated or down-regulated DEGs in the liver or brain tissues of Ggt7−/− mice vs. Ggt7+/+ mice

Article Snippet: Human hepatic cancer cell line HepG2, cervix cancer cell line HeLa, and HEK293FT cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Knock-Out, Western Blot, Derivative Assay, Control, RNA Sequencing, Functional Assay

Journal: Plants (Basel, Switzerland)

Article Title: Hydroethanolic Extract of Polygonum aviculare L. Mediates the Anti-Inflammatory Activity in RAW 264.7 Murine Macrophages Through Induction of Heme Oxygenase-1 and Inhibition of Inducible Nitric Oxide Synthase.

doi: 10.3390/plants13233314

Figure Lengend Snippet: Figure 4. Antioxidant effects of PAL hydroethanolic extract by activation of Nrf2 signaling path- way in RAW 264.7 macrophages. Protein levels of (A) nuclear Nrf2 and (B) cytoplasmic HO-1 in RAW 264.7 macrophages were quantitatively analyzed. (C) ARE activity by PAL hydroethanolic extract in HepG2-ARE cells. (D) Intracellular ROS level by PAL hydroethanolic extract in RAW 264.7 macrophages. Data are presented as the mean ± SEM (N = 3). A statistical significance com- pared with control group at p < 0.05 and p < 0.01 was marked by an asterisk (*) and double asterisk (**), respectively. SFN, sulforaphane; tBHP, tert-butyl hydroperoxide; tBHQ, tertiary-butylhydroquinone.

Article Snippet: HepG2 human hepatic cells harboring the construct containing the antioxidant response element (ARE) sequence following the luciferase-encoding gene were purchased from BPS Bioscience (#60513).

Techniques: Activation Assay, Activity Assay, Control