Journal: Nature Communications

Article Title: NAP-seq reveals multiple classes of structured noncoding RNAs with regulatory functions

doi: 10.1038/s41467-024-46596-y

Figure Lengend Snippet: a Schematic depiction of the NAP-seq method with the construction of TGS libraries and NGS libraries. Total RNA was extracted and pre-size selection of RNA fractions was performed by removing short RNAs. b The percentage of reads with at least one specific adapter in the NAP-seq-NGS libraries in humans and mice (3 biological replicates per sample). All data are plotted as the means ± SEMs. The number of reads (RPM, reads per million) for known ncRNAs was highly reproducible between replicates with both the NAP-seq-NGS ( c ) and NAP-seq-TGS ( d ) methods. p values were calculated by two-sided Pearson’s correlation test. e , f Coincidence analysis of both terminal sites in the NAP-seq-NGS reads and those in the known ncRNAs in HepG2 cells. The x -axis shows the distance from the annotated 5’-start ( e ) or 3’-end ( f ) site in the NAP-seq-NGS read to the corresponding terminal sites in the known ncRNAs, and the y -axis shows the number of reads (RPM) within a certain distance from the 5’-start or 3’-end site. The bottom panel shows a heatmap, in which each row represents a gene that matches the terminal sites with the NAP-seq-NGS read exactly, and each column represents the expression values of genes at a specific distance. expr, expression value. g Number of coincidences between the 5’-start sites in NAP-seq-TGS reads and the 5’-start sites in known ncRNAs in HepG2 cells. h Number of coincidences between the 3’-end sites in NAP-seq-TGS reads and the 3’-end sites in known ncRNAs in HepG2 cells. i Genome Browser visualization of NAP-seq 5’-start, 3’-end and coverage signals (RPM, reads per million), which were generated from NAP-seq-NGS and NAP-seq-TGS data, in a 5000-bp region of a human snoRNA cluster (chr17:7,573,750-7,578,750).

Article Snippet: To assay protein stability, HepG2 cells were treated with 0.05 mg/ml cycloheximide (MdBio, 66-81-9) for 15 min. For stress stimulation, poly (I:C) (InvivoGen, tlrl-pic) was transfected into HepG2 cells with Lipofectamine 2000 (Thermo Fisher, 11668019) at a final concentration of 2 μg/ml.

Techniques: Selection, Expressing, Generated

Journal: Nature Communications

Article Title: NAP-seq reveals multiple classes of structured noncoding RNAs with regulatory functions

doi: 10.1038/s41467-024-46596-y

Figure Lengend Snippet: a The number of coincident reads between the 5’-start site in NAP-seq-NGS reads and the 5’ splice sites in known introns in HepG2 cells. The x -axis shows the distance from the 5’-start sites of NAP-seq-NGS read to the annotated 5’ splice sites, and the y -axis shows the number of reads within a certain distance. The bottom panel shows a heatmap, in which each row represents an intron that shares the same 5’-start site with the NAP-seq-NGS read, and each column represents the intron expression value at a specific distance. expr, expression value. b The number of coincident reads between the 3’-ends in NAP-seq-NGS reads and the 3’ splice sites in known introns in HepG2 cells. c Top 10 enriched GO biological processes enriched with the host genes of 620 previously undiscovered sliRNAs identified in humans. p values were calculated by hypergeometric test. d Cumulative curves and box plots showing the lengths of sliRNAs identified by NAP-seq ( n = 620 examined over 17 independent experiments) and other introns ( n = 6951 according to the GENCODE), respectively. e Cumulative curves and box plots showing the average minimum free energy (avg. MFE) of sliRNAs and other introns with a length ≤500 nt in humans. p values in ( d ) and ( e ) were calculated by two-sided Mann–Whitney–Wilcoxon test. Each boxplot shows the minima, maxima, center, bounds of box, whiskers, first and third percentile. Examples of sliRNAs with highly stable secondary structures in humans ( f , g ) and mice ( h , i ). A negative z score indicated that a sequence was more stable than expected by chance. j The proposed model of sliRNA biogenesis. An intron lariat is spliced from the host gene and debranched to generate a linear intron. Then, the short linear intron may fold quickly and bind to RBPs (RNA binding proteins) to avoid degradation, contributing to its stability as a sliRNA in vivo. In contrast, long introns are degraded quickly by exonuclease digestion.

Article Snippet: To assay protein stability, HepG2 cells were treated with 0.05 mg/ml cycloheximide (MdBio, 66-81-9) for 15 min. For stress stimulation, poly (I:C) (InvivoGen, tlrl-pic) was transfected into HepG2 cells with Lipofectamine 2000 (Thermo Fisher, 11668019) at a final concentration of 2 μg/ml.

Techniques: Expressing, MANN-WHITNEY, Sequencing, RNA Binding Assay, In Vivo

Journal: Nature Communications

Article Title: NAP-seq reveals multiple classes of structured noncoding RNAs with regulatory functions

doi: 10.1038/s41467-024-46596-y

Figure Lengend Snippet: Statistics of the distances from napRNAs to annotated snoRNAs: start sites ( a ) and end sites ( b ). The x -axis shows the distance (nt) from napRNA 5’-start (or 3’-end) sites to snoRNA 5’-start (or 3’-end) sites, and the y -axis shows the number of napRNAs within the specified distance range. Genome Browser view of 5’-start, 3’-end and coverage signals (RPM, reads per million) in an extended region of two snotrons: hsa-snotron-1 ( c ) and mmu-snotron-5 ( d ). The highly stable secondary structures of snotron hsa-snotron-1 ( e ) and mmu-snotron-5 ( f ). g Verification of hsa-snotron-1 by irNorthern blotting in HepG2 cells. Source data are provided as a Source Data file. h Cumulative curves and box plots showing the distance from the host 3’-SS to the snoRNA 3’-end sites ( n = 35 examined over 17 independent experiments) identified by NAP-seq and to the other snoRNA 3’-end sites ( n = 363 according to the snoRNAbase). p value was calculated by two-sided Mann–Whitney–Wilcoxon test. Each boxplot shows the minima, maxima, center, bounds of box, whiskers, first and third percentile. i The proposed model of snotron biogenesis. An intron lariat is spliced from the host gene and is then debranched to generate a linear intron. Next, the RNA splicing intermediate is processed into a C/D box snoRNA or an H/ACA box snoRNA after the lariat intron is debranched (left path). Because the 3’-ends of shorter linear introns are formed into complex structures quickly and bind to RBPs, making the exonuclease unable to cut them, these intronic RNAs are stable as snotrons (right path). In addition, cellular stress might suppress snotron formation.

Article Snippet: To assay protein stability, HepG2 cells were treated with 0.05 mg/ml cycloheximide (MdBio, 66-81-9) for 15 min. For stress stimulation, poly (I:C) (InvivoGen, tlrl-pic) was transfected into HepG2 cells with Lipofectamine 2000 (Thermo Fisher, 11668019) at a final concentration of 2 μg/ml.

Techniques: MANN-WHITNEY

Journal: Nature Communications

Article Title: NAP-seq reveals multiple classes of structured noncoding RNAs with regulatory functions

doi: 10.1038/s41467-024-46596-y

Figure Lengend Snippet: a Statistics of the number of pre-miRNAs that intersected with the napRNAs. b Genome Browser view of the full-length misRNAs (RPM, reads per million) in an extended region of misRNA hsa-misRNA-4. The highly stable secondary structures of misRNAs hsa-misRNA-4 ( c ) and mmu-misRNA-18 ( d ). e , f The dynamic changes in misRNAs under different conditions or in different stages. Heatmaps showing the differentially expressed misRNAs in HepG2 cells under four stress-response conditions ( e ) and in C2C12 cells at four developmental stages ( f ). Each row represents a differentially expressed misRNA, and each column represents a treatment type. The color, ranging from blue to red, represents the relative expression value from low to high, respectively. g Genome Browser view of the processing of misRNAs in the miRNA-17-92 cluster. Genome Browser view of 5’-start, 3’-end and coverage signals (RPM, reads per million) in an extended region of the miRNA-17-92 cluster. h Diagram of miRNA processing from the miRNA cluster. The miRNA cluster pri-miR-17-92 simultaneously yielded five miRNA spacers. These miRNA spacers are shown in various colors based on the spacer type, which are the same as those for the TGS reads in ( g ). i The proposed model of misRNA biogenesis. During the miRNA maturation process in humans and mice, the spacer region between the two pre-miRNAs is separated and is then folded into a structural misRNA and bound by a microprocessor complex to prevent further degradation.

Article Snippet: To assay protein stability, HepG2 cells were treated with 0.05 mg/ml cycloheximide (MdBio, 66-81-9) for 15 min. For stress stimulation, poly (I:C) (InvivoGen, tlrl-pic) was transfected into HepG2 cells with Lipofectamine 2000 (Thermo Fisher, 11668019) at a final concentration of 2 μg/ml.

Techniques: Expressing

Journal: Nature Communications

Article Title: NAP-seq reveals multiple classes of structured noncoding RNAs with regulatory functions

doi: 10.1038/s41467-024-46596-y

Figure Lengend Snippet: a Evolutionary conservation analysis of the top 100 napRNAs (left) and the evolutionary conservation of DINAP in 100 vertebrates (right, conservation score calculated by PhyloP). b IGV plot showing the signal density of DKC1 binding in the region surrounding DINAP in HepG2 eCLIP data (ENCFF252TQP), Yellow columns represent the coverage of eCLIP enriched reads. c qPCR quantification (left) and western blotting analysis (right) of the nuclear and cytoplasmic distribution of DINAP and DKC1 in HepG2 cells. The subcellular distribution of MALAT1, SNORA23, pre-GAPDH and GAPDH RNA was assessed by qPCR. FBL, β-Tubulin and GAPDH in western blotting served as the nuclear and cytoplasmic markers, respectively. These genes were used as the positive controls. Protein loading in the western blotting was quantified by Coomassie staining. d In HepG2 WT cells and DKC1 knock-down cells respectively, subcellular localization of DINAP (red) is assessed by smiFiSH (the principle of smiFISH is shown on the top), and of DKC1, as assessed by immunofluorescence staining. Nuclear DNA was stained with DAPI. All images are representative of two biological replicates. e The interaction between DINAP and the DKC1 protein was confirmed by RIP-qPCR in lysates of wild-type HepG2 cells. f WB analysis of DKC1 protein levels in DINAP-overexpressing cells, KO-DINAP cells and KO-DINAP cells with restoration of DINAP expression treated with 0.05 mg/ml CHX. g The promotion of cell proliferation in DINAP-overexpressing cells and control cells was examined by a CCK-8 assay. h Effect of DINAP overexpression on the colony formation ability. Colonies were counted by ImageJ. i CCK-8 assays showing the effect of DKC1 on restoring cell proliferation in KO-DINAP cells. j Colony formation assay in wild-type HepG2 cells and KO-DINAP HepG2 cells with ectopic expression of DKC1. Representative images of crystal violet staining of cells in ( h ) and ( j ) are shown beside the histograms of colony numbers. Colonies were counted in three replicates. The values in ( c , e , g – j ) are mean ± SEM of three independent experiments. Two-sided t -test. Source data are provided as a Source Data file.

Article Snippet: To assay protein stability, HepG2 cells were treated with 0.05 mg/ml cycloheximide (MdBio, 66-81-9) for 15 min. For stress stimulation, poly (I:C) (InvivoGen, tlrl-pic) was transfected into HepG2 cells with Lipofectamine 2000 (Thermo Fisher, 11668019) at a final concentration of 2 μg/ml.

Techniques: Binding Assay, Western Blot, Staining, Knockdown, Immunofluorescence, Expressing, Control, CCK-8 Assay, Over Expression, Colony Assay

Journal: Pathogens

Article Title: A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

doi: 10.3390/pathogens11030355

Figure Lengend Snippet: Luciferase activity of four parental HEV replicons in comparison to mock-transfected control (H 2 O) and inactivated RdRp GAA mutant replicon based on p6LucA26. HepG2 cells were transfected with replicon ivtRNA by electroporation and seeded across four wells of a 96-well plate as technical quadruplicate. Replication was estimated by measuring total luciferase activity (relative light units; RLU) of each replicon. Error bars indicate standard deviation and asterisks indicate significance level (*** p ≤ 0.001; ** 0.001 < p ≤ 0.01; ns 0.05 < p ≤ 1). The data depict a representative experiment of three separate transfections.

Article Snippet: HepG2 cells were purchased from CLS (Eppelheim, Germany) and grown in DMEM (Gibco 52100, ThermoFisher, Waltham, MA, USA), supplemented with NaHCO 3 and Na-pyruvate, and 10% fetal bovine serum, including 10 mg/L gentamicin (PAN-Biotech, Aidenbach, Germany), and 250 mg/L amphotericin B (PAN-Biotech, Aidenbach, Germany) in cell culture flasks with vented caps (Corning, Corning, NY, USA) in a humidified incubator with 5% CO 2 .

Techniques: Luciferase, Activity Assay, Comparison, Transfection, Control, Mutagenesis, Electroporation, Standard Deviation

Journal: Pathogens

Article Title: A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

doi: 10.3390/pathogens11030355

Figure Lengend Snippet: ( A ) Structure of nine chimeric replicons that were constructed using p6LucA26 as backbone. Subgenomic fragments containing partial sequences of ORF1 (MYP, nucleotide positions 1–2139 in p6; VXH, nucleotide positions 2144–4059 in p6; RJ, nucleotide positions 4064–5347 in p6) were replaced with the corresponding sequences from HEV strains 47832mc, rab52, and rab81. Percentages annotated in the highlighted fragments represent pairwise amino acid sequence identity of the encoded proteins with the corresponding ones encoded by parental p6 replicon. Multiple amino acid sequence alignments of each ORF1-encoded protein segment are included in the . ( B ) HepG2 cells were transfected with replicon ivtRNA by electroporation and seeded across four wells of a 96-well plate as technical quadruplicates. Replication was estimated by measuring total luciferase activity (RLU) of each replicon. Error bars indicate standard deviation; asterisks indicate significance level in reference to p6LucA26 (**** p ≤ 0.0001; *** 0.0001 < p ≤ 0.001; ** 0.001 < p ≤ 0.01; * 0.01 < p ≤ 0.05; ns 0.05 < p ≤ 1). The data depict a representative experiment of three separate transfections. Note that the controls (p6LucA26, p6GAALucA26, and mock) are shown in each plot for illustrative purposes. The data depicted are derived from a single experiment with all chimeric replicons and one group of controls.

Article Snippet: HepG2 cells were purchased from CLS (Eppelheim, Germany) and grown in DMEM (Gibco 52100, ThermoFisher, Waltham, MA, USA), supplemented with NaHCO 3 and Na-pyruvate, and 10% fetal bovine serum, including 10 mg/L gentamicin (PAN-Biotech, Aidenbach, Germany), and 250 mg/L amphotericin B (PAN-Biotech, Aidenbach, Germany) in cell culture flasks with vented caps (Corning, Corning, NY, USA) in a humidified incubator with 5% CO 2 .

Techniques: Construct, Sequencing, Transfection, Electroporation, Luciferase, Activity Assay, Standard Deviation, Derivative Assay

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 1. Inhibition of HIF-hydroxylase activity by CoCl2. (a) In vitro prolyl hydroxylase activity assay. The GST-HIF1a-TADN fusion protein or the GST protein was incubated with HepG2 cell extract, cofactors, and [5-14C]2-oxoglutarate in the presence of CoCl2 (10 mM). The radioactivity associated to 14C-succinate was determined. In each experi- ment, the basal HIF-TADN-dependent activity (control) was set to 100% after being normalized by subtracting the GST-associated activity. Values are means ± SEM of three independent culture experiments. Statistics, Student’s t-test for paired values: *P £ 0.05 vs. control. (b) GST pull-down assay. HepG2 cells were treated with or without CoCl2 (10 mM). Cell extracts were prepared and incubated with the GST-HIF1a-TADN fusion protein supplemented with cofactors. Glutathione-Sepharose beads and [35S]VHL were then added and the bound VHL was recovered, subjected to SDS–PAGE, and visualized by phosphoimaging. The input remains from directly loaded [35S]VHL. The two bands represent the 213 and 160 amino acid VHL translation products (105, 106).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Activity Assay, In Vitro, Incubation, Radioactivity, Control, Pull Down Assay, SDS Page

Journal: Methods in Molecular Biology

Article Title: Transcription Factors

doi: 10.1007/978-1-60761-738-9

Figure Lengend Snippet: Fig. 2. Inhibition of the IGF-1-mediated HIF-1alpha induction by the PI(3)-kinase inhibitor LY294002 and the MEK inhibitor U0126. Serum-starved HepG2 cells were pretreated with 10 µM LY294002 or 10 µM U0126 for 30 min and then treated either with or without 100 nM human IGF-1 (Sigma) and exposed to normoxia (16% O2) or hypoxia (8% O2) for 4 h. Acetic acid was used in controls at a final concentration of 100 nM to keep the pH constant. 100 µg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1. Autoradiographic signals were detected by chemiluminescence (77).

Article Snippet: 100 μg of protein from HepG2 cell lysates were analyzed by Western Blotting with antibodies against HIF-1alpha (Novus Biological Transduction Lab, 1:2,000), or against phospho-ERK1/2 (cell signaling, 1:1,000) where HepG2 cells were stimulated for 15 min with IGF-1.

Techniques: Inhibition, Concentration Assay, Western Blot, Transduction

Journal: Journal of Diabetes Research

Article Title: Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes

doi: 10.1155/2019/9582714

Figure Lengend Snippet: Triglycerides and diacylglycerol are increased by HFD. (a–d) Oil Red O staining showed a marked increase in fat droplets in mouse hepatic tissue following high fat (c) and high fat with carrageenan (d) with a little sign of fat droplets in the carrageenan-treated (b) or control hepatic tissue (a). Scale bar = 50 μ m. (e) Triglycerides were measured in the hepatic and pancreatic tissues of the study mice and increased following HFD but not carrageenan ( p < 0.001, n = 12). (f) DAG levels in the hepatic and pancreatic tissues increased following HFD in the mice but not following carrageenan exposure ( p < 0.001, n = 12). (g) Palmitic acid increased DAG in the HepG2 cells ( p < 0.001, n = 3). CGN = carrageenan; DAG = diacylglycerol; PA = palmitic acid.

Article Snippet: Diacylglycerol (DAG) concentration was determined in tissue homogenates and in HepG2 cell extracts by a competitive enzyme immunoassay, as per directions (Aviva Systems Biology, San Diego, CA).

Techniques: Staining, Control

Journal: Journal of Diabetes Research

Article Title: Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes

doi: 10.1155/2019/9582714

Figure Lengend Snippet: Serum galectin-3 increased following carrageenan or HFD. (a) Serum galectin-3 increased following carrageenan or HFD or their combination ( p < 0.001, n = 18). (b) Galectin-3 binding with the insulin receptor increased in the liver and muscle membrane preparations of the treated mice, with the greatest effect following the combined exposure ( p < 0.001, n = 12). (c) In HepG2 cells, the insulin-induced increase in phospho(Tyr)-IRS1 was significantly inhibited by administration of exogenous recombinant human galectin-3 ( p < 0.001, n = 3). (d) The insulin-induced glucose uptake in the HepG2 cells was blocked by administration of exogenous recombinant human galectin-3 ( p < 0.001, n = 3). (e) The Pearson correlation r between the glucose uptake and the phospho(Tyr)-IRS-1 in the HepG2 cells was 0.985. (f) Activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase), which removes 4-sulfate groups from the nonreducing end of chondroitin 4-sulfate and dermatan sulfate, was inhibited by exposure to carrageenan, but not by the HFD, in the liver and pancreas of the treated mice ( p < 0.001, n = 12). ARSB = arylsulfatase B; CGN = carrageenan; IRS = insulin receptor substrate.

Article Snippet: Diacylglycerol (DAG) concentration was determined in tissue homogenates and in HepG2 cell extracts by a competitive enzyme immunoassay, as per directions (Aviva Systems Biology, San Diego, CA).

Techniques: Binding Assay, Membrane, Recombinant, Activity Assay

Journal: Nature Communications

Article Title: Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

doi: 10.1038/s41467-022-35515-8

Figure Lengend Snippet: a The cytotoxicity study of Cu@SA, Au@SA, and Au/Cu 0 @SAnanocubes with HepG2-Red-FLuc liver cancer cells by using MTT assay through 100 ppm in Cu concentration. b Live (green color) and dead cells (red color) stained with fluorescent green dye (Calein-AM) and red dye (propidium iodide), respectively, for cancer cells treated with Cu@SA, Au@SA, and Au/Cu 0 @SA nanocubes (one representative data was shown from three independently repeated experiments). c The profiles of cells viability with different Au 0.02 Cu 0.98 @SA concentrations for 24 h incubation at 37 °C. d Flow cytometry analysis of HepG2-Red-FLuc cancer cells treated with Cu@SA, Au@SA, and Au/Cu 0 @SA nanocubes. All data were obtained in triplicate ( n = 3, the error bars represented mean ± SD). Source data are provided as a Source Data file.

Article Snippet: HepG2-Red-FLuc cells were obtained from PerkinElmer (Product No. BW134280).

Techniques: MTT Assay, Concentration Assay, Staining, Incubation, Flow Cytometry

Journal: Nature Communications

Article Title: Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

doi: 10.1038/s41467-022-35515-8

Figure Lengend Snippet: a The morphology of Au 0.02 Cu 0.98 @SA nanocubes treated with or without HepG2-Red-FLuc cancer cells as a function of time. b Cu + release stained by CopperGreen dye showing green color as a function of time and the corresponding quantitative analysis. c H 2 O 2 generation stained by hydrogen peroxide assay kit showing green color as a function of time and the corresponding quantitative analysis. d •OH generation stained by APF dye showing green color as a function of time and the corresponding quantitative analysis. e Analysis of hemolysis in blood containing 2% red blood cells from Au 0.02 Cu 0.98 @SA nanocubes. Negative and positive controls were conducted by immersing red blood cells in PBS and water, respectively. f Cytotoxicity analysis of vascular endothelial cells treated with Au 0.02 Cu 0.98 @SA nanocubes. The scale bar is 100 μm in all fluorescence images. All data were obtained in triplicate ( n = 3, the error bars represented mean ± SD, and p -values were calculated by one-way ANOVA). Source data are provided as a Source Data file (one representative data was shown from three independently repeated experiments).

Article Snippet: HepG2-Red-FLuc cells were obtained from PerkinElmer (Product No. BW134280).

Techniques: Staining, H2O2 Assay, Fluorescence

Journal: Nature Communications

Article Title: Atomically dispersed golds on degradable zero-valent copper nanocubes augment oxygen driven Fenton-like reaction for effective orthotopic tumor therapy

doi: 10.1038/s41467-022-35515-8

Figure Lengend Snippet: a Histology morphology of each organ with PBS and Au 0.02 Cu 0.98 @SA nanocubes on post-injection day 7 was observed by H&E staining (scale bar, 200 µm). b The biodistribution was determined by Cu concentration collected from Au 0.02 Cu 0.98 @SA nanocubes through intravenous injection (the inset showing accumulation without liver, n = 3). c Antitumor efficacy of different nanocubes (Au@SA, Au 0.5 Cu 0.5 @SA, Cu@SA, and Au 0.02 Cu 0.98 @SA) in HepG2-Red-FLuc orthotopic liver tumor mice ( n = 3). Tumor growth was monitored by the IVIS system. d The IVIS bioluminescence of livers with hepatocellular carcinoma in each treatment group after mice were sacrificed ( n = 3). e The expression of phospho-histone H2A.X (Ser139) and cleaved caspase-3 (Asp175) within hepatocellular carcinoma from each treatment group mice by IHC staining (scale bar, 100 µm). Experiments of H&E staining and IHC staining were repeated at least three times independently with similar tendencies and the result from a representative experiment was shown. The error bars represented mean ± SEM ( b–d ). The p -value was calculated by one-way ANOVA.

Article Snippet: HepG2-Red-FLuc cells were obtained from PerkinElmer (Product No. BW134280).

Techniques: Injection, Staining, Concentration Assay, Expressing, Immunohistochemistry

Journal: Endocrinology

Article Title: Endogenous IGFBP-3 Mediates Intrinsic Apoptosis Through Modulation of Nur77 Phosphorylation and Nuclear Export.

doi: 10.1210/en.2015-1215

Figure Lengend Snippet: Figure 1. Endogenous Nur77 is knocked down using two different siRNA oligos and is detectable in MAC-T cells with a commercially available antibody. Cells were transfected with scramble (SCR) or Nur77 siRNA (Oligo No. 1 or Oligo No. 2). Total RNA (A) or protein (B) was isolated from WCLs after 48 h and analyzed for Nur77 by RT-qPCR (corrected for cyclophilin) or Western immunoblot (50 g protein), respectively. For the immunoblot, HSP60 served as a loading control. Bars represent mean SE of three experiments. C, To confirm specificity of the Nur77 antibody, MAC-T WCL (50 g), WCL from MAC-T cells transfected with myc-tagged Nur77 (3 g), HepG2 WCL (50 g), and nuclear fractions from MAC-T cells (5 g) were immunoblotted for Nur77 on one half of the membrane and myc on the other half. Actin served as a loading control.

Article Snippet: HepG2 cell lysate was obtained from Novus Biologicals.

Techniques: Transfection, Isolation, Quantitative RT-PCR, Western Blot, Control, Membrane

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on hepatic apoC-III expression. Rat primary hepatocytes were transduced with Foxo1 or LacZ vector at an MOI of 50 PFU/cell or mock-transduced with PBS. After 24 hours of transduction, the intracellular levels of apoC-III (A), Foxo1 (B), and GK (C) mRNA were determined by real-time RT-PCR using β-actin mRNA as control. The effect of Foxo1 on hepatic apoC-III expression in response to insulin was assayed in HepG2 cells. Cells were transduced with Foxo1, Foxo1-ADA, or control LacZ vector (50 PFU/cell) in the absence or presence of insulin at different concentrations. Twenty-four hours after transduction, cells were collected for determination of the intracellular levels of apoC-III mRNA induced by Foxo1 (D) and Foxo1-ADA (E). *P < 0.05, **P < 0.005; significantly different from controls. NS, not significant by ANOVA. Data were from 3 independent experiments.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Expressing, Transduction, Plasmid Preparation, Quantitative RT-PCR

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Effects of Foxo1 on the human APOC3 promoter activity. (A) The APOC-III promoter–directed luciferase reporter system. The wild-type and mutant IRE sequences are underlined. (B) Foxo1-mediated induction of the APOC3 promoter activity. HepG2 cells were transfected by pHD317 together with Foxo1 construct, or with both Foxo1 and Foxo1-Ø256 constructs. For each construct, 1 μg of DNA for each construct was used in transfection. For normalization of transfection efficiency, 1 μg pCMV5-LacZ DNA was included for normalization of transfection efficiency. (C) The APOC3 promoter variants in the luciferase reporter system. (D) Responses of APOC3 promoter variants to Foxo1 production. HepG2 cells were transfected with individual test plasmids in the absence (–) or presence (+) of pCMV5-Foxo1. The relative luciferase activity, after normalizing to β-gal activity, was compared between basal (–) and Foxo1-inducible (+) conditions. (E) Responses of wild-type and mutant APOC3 promoters to insulin. Test plasmids were transduced into HepG2 cells in the presence and absence of pCMV5-Foxo1 transfection in culture media, either supplemented with or without insulin (30 nM). The relative luciferase activity in transduced cells was determined using β-gal activity as control. *P < 0.001 vs. controls.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: Activity Assay, Luciferase, Mutagenesis, Transfection, Construct

Journal:

Article Title: Foxo1 mediates insulin action on apoC-III and triglyceride metabolism

doi: 10.1172/JCI200419992

Figure Lengend Snippet: Molecular interaction between Foxo1 and the APOC3 promoter. Molecular association between Foxo1 and the APOC3 promoter was analyzed by EMSA and ChIP. Aliquots of Foxo1 protein from linked in vitro transcription-translation products (5 μg) were incubated with 2.5 μl of radioactively labeled DNA corresponding to –467/–440 nt in the human APOC3 promoter (WT-IRE) (A), a mutant APOC3 IRE (mt-IRE) containing 2 substitutions, of –A458C and –A460G, and a control PEPCK IRE DNA (B), followed by electrophoresis through 8% nondenaturing polyacrylamide gels for 30 minutes. Lane 1, DNA probe alone. Lane 2, DNA probe + Foxo1 protein lysates. Lane 3, DNA probe + Foxo1 protein lysates + anti-Foxo1 antibody (1 μg). Lane 4, DNA probe + Foxo1 protein lysates + nonlabeled competitor DNA at a molar concentration of 50-fold excess. Free, shifted, and supershifted DNA bands were visualized by autoradiography. For ChIP assay, HepG2 cells were transduced with Foxo1 vector at an MOI of 50 PFU/cell. Cells were harvested 24 hours later and subjected to ChIP using PBS as a negative control (lane 5), control IgG (lane 6), and anti-Foxo1 antibody (lane 7). The coimmunoprecipitated chromatin DNA was analyzed by immunoblot (C) using anti-Foxo1 antibody and PCR (D) using the primers that correspond to –655/–20 nt of the APOC3 promoter.

Article Snippet: As controls, aliquots (1 × 10 6 ) of Foxo1 vector–transduced HepG2 cells were treated identically for the preparation of cell lysates, which were immunoprecipitated with 5 μg of control IgG (sheep IgG, Rockland Immunochemicals) or PBS buffer.

Techniques: In Vitro, Incubation, Labeling, Mutagenesis, Electrophoresis, Concentration Assay, Autoradiography, Transduction, Plasmid Preparation, Negative Control, Western Blot