Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 1. Interaction and colocalization of SMILE with ERR. A, in vitro GST pulldown assays. 35S-Radiolabeled ERR, ERR, or ERR proteins were incubated with GST or GST-SMILE fusion proteins. The input lane repre- sents 10% of the total volume of in vitro-translated proteins used for bind- ing assay. Protein interactions were detected via autoradiography. B, in vivo interaction between exogenous ERR and SMILE. HepG2 cells were cotrans- fected with pSG5-HA-ERR and pEBG-SMILE (GST-SMILE) or pEBG alone (GST). Protein interactions were examined via in vivo GST pulldown. The top and middle panels (GST puri) show GST beads-precipitated HA-ERR and GST fusion proteins, respectively. The bottom panel shows the protein expression levelsofHA-ERRincelllysates.C,invivointeractionofendogenousERRand SMILE. Coimmunoprecipitation assays were performed using cell extract from HepG2 cells, mouse liver, kidney, and heart tissues with anti-SMILE anti- body.EndogenousSMILEwasimmunoprecipitated(IP)withERR(upperpan- els). The proteins in the cell lysates (middle and lower panels) were analyzed with Western blot (WB) analysis using indicated antibodies. D, colocalizations of SMILE with ERR. HeLa cells were transfected with 0.1 g of expression vectors encoding GFP-SMILE and HA-ERR. HA fusion proteins were detected with dye Alexa 594-conjugated anti-HA monoclonal antibody. The cell images were captured under 400 magnifications. The data shown are rep- resentative of at least three independent experiments. DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: In Vitro, Incubation, Autoradiography, In Vivo, Expressing, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 2. Effect of overexpression and knockdown of SMILE on ERR transactivation. Reporter assays (A–D) were performed as described under “Experimental Procedures.” A and B, effect of SMILE on ERR-mediated tran- scriptional activity. 293T and HepG2 cells were cotransfected with 0.3 g of pcDNA3-FLAG-ERR, and 0.1 g of sft4-Luc reporter vectors, together with indicated amounts of plasmids expressing WT SMILE, SMILE-L (SMILE-83Leu), and SMILE-S (SMILE-1Phe). C and D, siSMILE increases ERR transactivation. 293T and HepG2 cells were transfected with pSUPER (control (con)), or pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2), or pSUPER siSHP (siSHP). After 24 h, the cells were cotransfected with expression vector for FLAG-ERR and sft4-Luc reporter vectors. The luciferase activity was meas- ured 48 h after the second transfection. The means S.D. (n 3) of a repre- sentative experiment are shown. **, p 0.01, using Student’s t test. E, effects of overexpressed SMILE on the protein levels of FLAG-ERR. 293T cells were cotransfected with various plasmids as indicated. The proteins of FLAG-ERR, SMILE, and tubulin were detected by respective antibodies though Western blot analysis. F, effect of siRNAs for SMILE or SHP on the expression of SMILE and SHP. 293T and HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER (Con), and after 72 h the total RNA was isolated. The mRNA levels of SHP and SMILE were measured via RT-PCR analysis, with -actin shown as a control. The data shown are repre- sentative of at least three independent experiments.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: Over Expression, Knockdown, Activity Assay, Expressing, Transfection, Control, Plasmid Preparation, Luciferase, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 3. Interaction domains of ERR and SMILE. A, schematic representation of the structures of ERR mutants. AF1, activation function-1 domain; DBD, DNA binding domain. The numbers in the figure indicate the amino acid (aa) residues. B, SMILE interacts with the AF2 domain of ERR. 35S-Radiolabeled ERR or ERRAF2 proteins were incubated with GST or GST-SMILE fusion proteins. Protein interactions were detected via auto- radiography.C,schematicrepresentationofthestructuresofSMILEmutants.Thenumbersinthefigureindicate the amino acid residues. D, in vivo interaction assays between ERR and SMILE mutants. HepG2 cells were cotransfected with expression vectors for HA-ERR and pEBG alone (GST) or pEBG-SMILE (GST-SMILE) fusions as indicated. Protein interactions were examined via in vivo GST pulldown. The top and middle panels (GST puri) show GST bead-precipitated HA-ERR and GST fusions, respectively. The bottom panel shows the protein expression levels of HA-ERR in cell lysates. wt, wild-type. The data shown are representative of at least three independent experiments with similar results.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: Activation Assay, Binding Assay, Incubation, In Vivo, Expressing

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 4. Competition between SMILE and coactivators. Reporter assays in A–C (upper panels) were performed asdescribedunder“ExperimentalProcedures.”ThemeansS.D.(n3)ofarepresentativeexperimentareshown. HepG2 cells were cotransfected with 0.1 g of sft4-Luc reporter plasmids, together with the indicated expression vectors for FLAG-ERR (0.2 g), FLAG-SMILE, HA-PGC-1, HA-PGC-1, and HA-GRIP1. A–C, lower panels, in vitro competition of SMILE with PGC-1, HA-PGC-1, or HA-GRIP1. 35S-Radiolabeled ERR were incubated with the indi- catedGSTorGST-SMILEfusionproteinstogetherwithincreasingamountsofunlabeledinvitro-translatedHA-PGC- 1,HA-PGC-1,orHA-GRIP1(0,3,6,or12l)proteins.Theproteininteractionsweredetectedviaautoradiography. D,schematicrepresentationofSMILEandLXXLLmotifmutantsofSMILE.Upperarrowsindicatethelocationsoffour LXXLL motifs in SMILE and lower arrows indicate the mutation of LXXLL motifs to AXXAL. m1, SMILE-m1 (1st LXXLL mutatedtoAXXAL);m2,SMILE-m2(2ndLXXLLmutatedtoAXXAL);m3,SMILE-m3(3rdLXXLLmutatedtoAXXAL);m4, SMILE-m4 (4th LXXLL mutated to AXXAL); m5, SMILE-m5 (all of the four LXXLL mutated to AXXAL). E, Western blot analysis using specific antibodies for SMILE and tubulin, with whole-cell extracts from HepG2 cells transfected with expression vectors encoding wild-type (wt) FLAG-SMILE or FLAG-SMILE-m1, -m2, -m3, -m4, and -m5. F, effects of SMILE LXXLL mutants on ERR-mediated transcriptional activity. HepG2 cells were cotransfected with reporter vectorsft4-Luc,togetherwithindicatedexpressionvectorforERR,wild-type(wt)FLAG-SMILEorFLAG-SMILELXXLL mutants. Luciferase activity was measured after 48 h of transfection. The means S.D. (n 3) of a representative experiment are shown. G, in vivo interactions of SMILE LXXLL mutants with ERR. HepG2 cells were cotransfected with expression vectors for FLAG-ERR and wild-type pEBG-SMILE (GST-SMILE), or indicated GST-SMILE mutants, or pEBGalone(GST).ProteininteractionswereexaminedviainvivoGSTpulldown.Thetopandmiddlepanels(GSTpuri) showGSTbead-precipitatedFLAG-ERRandGSTfusions,respectively.Thebottompanelshowstheproteinexpres- sionlevelsofFLAG-ERRincelllysates.Thedatashownarerepresentativeofatleastthreeindependentexperiments with similar results.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Incubation, Mutagenesis, Western Blot, Transfection, Activity Assay, Luciferase, In Vivo

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 5. Involvement of SIRT1 in SMILE repression of ERR. Reporter assays in A–D, H, and I were per- formed as described under “Experimental Procedures.” The means S.D. (n 3) of a representative experi- mentareshown.HepG2cellswerecotransfectedwith0.1gofindicatedreporterplasmids,(HNF4)8-Luc(A),or sft4-Luc (B–D) and 0.1 g of pcDNA3-HA-HNF4 (A) or pcDNA3-FLAG-ERR (B–D), together with or without pcDNA3-FLAG-SMILE (0.2 g in A–C, and 0.1 g in D), and indicated doses of pcDNA3-Myc-SIRT1 or -SIRT1H363Y. 36 h after transfection, the cells in A–C were left untreated or treated with HDAC inhibitor TSA or SIRT1inhibitorssirtinol(20M),EX527(10M),ornicotinamide(NAM,20mM)for12hpriortothemeasurement of luciferase activity. E, in vitro interaction of SMILE and SIRT1. 35S-Radiolabeled SIRT1 protein was incubated with GST or GST-SMILE or GST-ERR fusion proteins. Protein interactions were detected via autoradiography. F, in vivo interaction of exogenous SIRT1 and SMILE. HepG2 cells were cotransfected with wild-type pcDNA3-myc-SIRT1 or pcDNA3-myc-SIRT1H363Y and pEBG-SMILE (GST-SMILE) or pEBG alone (GST). Protein interactions were examined via in vivo GST pulldown. The top and middle panels (GST puri) show GST bead- precipitated Myc-SIRT1 and GST fusions, respectively. The bottom panel shows the protein expression levels of Myc-SIRT1 in cell lysates. G, in vivo interaction of endogenous SIRT1 and SMILE. Coimmunoprecipitation assays were performed with cell extract from HepG2 cells using anti-SMILE antibody. Endogenous SMILE was immu- noprecipitatedwithSIRT1(upperpanel).Theproteinsinthecelllysates(middleandlowerpanels)wereanalyzed by Western blot (WB) analysis using indicated antibodies. H and I, HepG2 cells were transfected with pSUPER (control (Con)) or pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2), or pSUPER siSIRT1 (siSIRT1). After 24 h, the cells were cotransfected with expression vector for FLAG-ERR and sft4-Luc reporter vectors. 36 h after the second transfection, the cells were treated with or without indicated SIRT1 activators Resveratrol (100 nM) or piceatannol (20 M) for 12 h prior to the measurement of luciferase activity. J, effect of siSMILE and siSIRT1 on the expression of SMILE and SIRT1. HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER (Con), and after 72 h the total RNA was isolated. The mRNA levels of SHP and SMILE were measured via RT-PCR analysis with -actin shown as a control. The data shown are represent- ative of at least three independent experiments.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: Transfection, Luciferase, Activity Assay, In Vitro, Incubation, Autoradiography, In Vivo, Expressing, Western Blot, Control, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation

doi: 10.1074/jbc.m109.034165

Figure Lengend Snippet: FIGURE 7. Autoregulatory loop controlling SMILE gene expression by ERR. A, upper panel shows the relative ERR and SMILE mRNA levels analyzed by quantitative real time PCR (standardized using -actin). Normalized basal levels of each transcript were assigned an arbitrary value of 1.0 for comparison. Lower panel showstheproteinexpressionlevelsofERRandSMILE.Tubulinwasusedasaloadingcontrol.HepG2cellswere infected with adenovirus vector (Ad-Null and Ad-ERR) at indicated multiplicity of infection (0, 50, or 100). B, ERR activates human SMILE promoter activity. HepG2 cell were cotransfected with 0.1 g of SMILE-Luc reporter vector and indicated amount of expression vectors encoding ERR, ERR, or ERR. C, SMILE inhibits ERR-mediated and PGC-1-enhanced SMILE promoter activity. HepG2 cells were cotransfected with 0.2 g of SMILE-Luc reporter together with or without 0.2 g of ERR expression vector and indicated amount of expres- sion plasmids for SMILE and PGC-1. D, schematic representation of wild-type and mutant hSMILE promoter constructs. The putative ERR binding sites are shown, and the mutated ERRE is indicated with X. E, ERRE2 is essential for the activation of SMILE promoter by ERR. HepG2 cells were cotransfected with 0.2 g of wild-type or mutant SMILE promoter constructs along with or without 0.2 g of ERR expression vector. Luciferase activity was measured 48 h after transfection. F, ERR binds to SMILE promoter in ChIP assays. HepG2 cells were transfected with expression vector for HA or HA-ERR. Chromatin fragments were prepared from the trans- fected cells and immunoprecipitated with anti-HA antibody or unrelated immunoglobin G (IgG) as indicated. DNA fragments covering ERRE (996 to 735 bp) on SMILE promoter and a control region (220 to 19 bp) were PCR-amplified. Data shown are representative of three experiments. G, schematic representation of the autoregulatory loop controlling the expression of SMILE by ERR and the mechanisms of SMILE repression on ERR, recruitment of SIRT1 and dissociation of coactivator PGC-1.

Article Snippet: Cell Culture, Transient Transfection Assay, and Luciferase Assay—HEK293T (293T, human embryonic kidney), HepG2 (human hepatoma), and HeLa cells (cervical cancer) were obtained from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Comparison, Infection, Plasmid Preparation, Activity Assay, Expressing, Mutagenesis, Construct, Binding Assay, Activation Assay, Luciferase, Transfection, Immunoprecipitation, Control, Amplification

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism

doi: 10.1152/ajpgi.00322.2010

Figure Lengend Snippet: Nrf2 induction of SHP gene expression. A: semiquantitative PCR analysis of gene expression using total RNA isolated from primary hepatocytes of 2-mo-old male wild-type (+, lane 1) and Nrf2−/− (−, lane 2) mice, or from Nrf2−/− hepatocytes transfected with a pcDNA3 control (pc, lane 3) or a Nrf2 expression plasmid (Nrf2, lane 4) that expresses a full length mouse Nrf2. B: real-time PCR analysis of gene expression in primary hepatocytes of 2-mo-old male wild-type mice overexpressed with control green fluorescent protein (GFP; GFPAd) or Nrf2 adenovirus (Nrf2Ad). Nqo1, a Nrf2 target gene, was used as a positive control. C: real-time PCR analysis of gene expression in primary hepatocytes of 2-mo-old male wild-type mice treated with oxidized free fatty acid DHA (oxDHA, 50 μm, 5 h). D: real-time PCR analysis of gene expression in mouse hepatocyte Nmuli cells treated with 0.1% butylated hydroxyanisole (BHA; dissolved in DMSO) for 5 h. E: real-time PCR analysis of liver gene expression in 2-mo-old male wild-type mice fed with 0.5% BHA for 2 wk (n = 5/group). F: real-time PCR analysis of gene expression in Nrf2+/+ and Nrf2−/− hepatocytes treated with 0.1% BHA for 5 h. G: real-time PCR analysis of liver gene expression in 2-mo-old male Nrf2+/+ and Nrf2−/− mice fed with 0.5% BHA for 2 wk. B–F used HPRT and F–G used β-actin as an internal control. H: Nrf2−/− hepatocytes were transduced with GFP control or SHP adenovirus and the total TG and cholesterol (Chol) contents were determined. I: luciferase (Luc.)/β-galactosidase (β-gal, gal.) activities (act.) determined in HEK293 cells transfected with the SHP promoter reporter (∼2 kb) in the presence of Nrf2 (200 ng) expression plasmid and its coactivators (200 ng each). The β-gal expression vector (100 ng) was cotransfected as a control for transfection efficiency. Luciferase activity was normalized against β-gal activity. J: chromatin immunoprecipitation (ChIP) assays of Nrf2 coimmunoprecipitation (CoIP) on the SHP promoter region containing a conserved antioxidant response element (ARE) by using specific Nrf2 antibodies. Nrf2 expression vector was cotransfected with the mouse SHP promoter in HepG2 cells for 2 days and the cells were harvested. Rabbit anti-Nrf2 antibody (5 μg) was used for the CoIP. Rabbit IgG was used as a negative control. The fragment targeting for the ARE was amplified by PCR. The primers designed at the distal site of SHP promoter (−5 kb) was set as a negative control. The location of ARE and primers used for ChIP assays is presented in Fig. S2. Data (B–F) are represented as means ± SE of triplicate assays. Ad, adenovirus; *P < 0.01.

Article Snippet: For chromatin immunoprecipitation (ChIP) assays, HepG2 cells were used: chromatin was cross-linked and immunoprecipitated with rabbit anti-Nrf2 antibodies (sc-13032, Santa Cruz) and rabbit normal IgG (serving as a negative control).

Techniques: Gene Expression, Isolation, Transfection, Control, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Positive Control, Transduction, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Negative Control, Amplification

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Metabolism of Beclomethasone Dipropionate by Cytochrome P450 3A Enzymes

doi: 10.1124/jpet.112.202556

Figure Lengend Snippet: (A) Quantification of BDP metabolites produced by A549 cells treated with BDP or BDP with esterase inhibitors (+EI). (B) Relative quantification of BDP metabolites produced by DPX2 cells treated with BDP or BDP with esterase inhibitors (+EI). Data are the mean and standard deviation from six replicates. n.d. Signifies that the metabolite was not detected. (C and D) CYP3A enzyme mRNA abundance, measured by qPCR in A549 (C) and DPX2 (D) cells. Data are represented as the number of mRNA copies per 10,000 copies of β2-macroglobulin (a “housekeeping” gene). Statistics used for A549 cell data analysis were one-way analysis of variance with Dunnett’s post-hoc test. For DPX2 cell data analysis two-way ANOVA with Bonferronni post-hoc testing was used. Data are the mean and standard deviation from 6 replicates. n.d. Signifies that mRNA was not detected. *P < 0.05; ***P < 0.001; ****P < 0.0001.

Article Snippet: DPX2 cells (HepG2 background with human PXR stably overexpressed to drive the expression of a CYP3A4 reporter gene construct by PXR agonists) were provided by Dr. Judy Raucy (Puracyp Inc., Carlsbad, CA).

Techniques: Produced, Quantitative Proteomics, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Circular RNA Circ_0000098 Elevates ALX4 Expression via Adsorbing miR-1204 to Inhibit the Progression of Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.696078

Figure Lengend Snippet: Knocking down circ_0000098 promoted the proliferation, migration, and invasion of HCC cells. (A) qRT-PCR was used to detect the expression of circ_0000098 in Huh7 and SMMC-7721 cells after the transfection of si-circ_0000098#1 and #2. (B) CCK-8 assay was used to evaluate cell proliferation. (C) The migration ability of cells was detected by wound healing assay (Scale bar, 100 μm). (D, E) Transwell assay was used to detect cell migration and invasion (Scale bar, 250 μm). (F) Western blot assay was used to detect the expression levels of E-cadherin, N-cadherin, and Vimentin in Huh7 and SMMC-7721 cells with down-regulated circ_0000098 expression. ** P < 0.01 and *** P < 0.001.

Article Snippet: Human HCC cell lines (HepG2, SNU423, and SNU475) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Migration, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Frontiers in Oncology

Article Title: Circular RNA Circ_0000098 Elevates ALX4 Expression via Adsorbing miR-1204 to Inhibit the Progression of Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.696078

Figure Lengend Snippet: MiR-1204 was the target of circ_0000098. (A) qRT-PCR was used to detect the expressions of circ_0000098, U6, and GAPDH mRNA in the nucleus and cytoplasm of HCC cells. (B) The binding sequence between miR-1204 and circ_0000098 was predicted by Circular RNA Interactome database. (C) Dual-luciferase reporter assay was used to verify the binding relationship between miR-1204 and circ_0000098. (D) The direct interaction between circ_0000098 and miR-1204 was determined by RIP assay. (E) qRT-PCR was used to detect the expression of miR-1204 in cells transfected with si-circ_0000098#1 and #2. (F, G) The expression of miR-1204 in HCC tissues and cells was detected by qRT-PCR. (H) Pearson’s correlation analysis was used to analyze the correlation between miR-1204 and circ_0000098 expressions in 34 HCC tissues. ** P < 0.01 and *** P < 0.001.

Article Snippet: Human HCC cell lines (HepG2, SNU423, and SNU475) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Reporter Assay, Expressing, Transfection

Journal: Frontiers in Oncology

Article Title: Circular RNA Circ_0000098 Elevates ALX4 Expression via Adsorbing miR-1204 to Inhibit the Progression of Hepatocellular Carcinoma

doi: 10.3389/fonc.2021.696078

Figure Lengend Snippet: Circ_0000098 regulated the progression of HCC by adsorbing miR-1204. HCC cells were transfected with si-NC+miR-in, si-NC+miR-1204-in, si-circ_0000098#1+miR-in, or si-circ_0000098#1+miR-1204-in. (A) The expression of miR-1204 in Huh7 and SMMC-7721 cells was detected by qRT-PCR. (B) The cell proliferation was detected by CCK-8 assay. (C) The migration ability of cells was detected by wound healing assay. (D, E) Transwell assay was used to detect cell migration and invasion. (F) Western blot assay was used to detect the expression levels of E-cadherin, N-cadherin, and Vimentin in Huh7 and SMMC-7721 cells. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human HCC cell lines (HepG2, SNU423, and SNU475) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Bioscience Reports

Article Title: Promoter analysis of the DHCR24 (3β-hydroxysterol Δ 24 -reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

doi: 10.1042/BSR20120095

Figure Lengend Snippet: HepG2 cells were incubated with lovastatin (Lov, 10 μM) or LDL (30 μg of cholesterol/ml) in a lipoprotein deficient medium. Samples were harvested after 24 h and the expression of DHCR24 ( A ) and HMGCR ( B ) was measured by real-time RT–PCR using the relative quantification method by comparison with the expression of the housekeeping gene RPLP0 . The results are expressed as the relative amount of mRNA compared with the level under control conditions. Results are the means±S.E.M. for three independent experiments. * P <0.05 and ** P <0.01 by the paired Student's t test.

Article Snippet: The promoter region of the human DHCR24 gene was generated by PCR amplification using HepG2 cell genomic DNA as a template and Platinum® Pfx DNA Polymerase (Invitrogen).

Techniques: Incubation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Comparison, Control

Journal: Bioscience Reports

Article Title: Promoter analysis of the DHCR24 (3β-hydroxysterol Δ 24 -reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

doi: 10.1042/BSR20120095

Figure Lengend Snippet: A fragment of 1018 bp of the 5′ flanking region containing the promoter of DHCR24 was cloned into the pGL3-basic vector as described in the Experimental section. Unidirectional serial deletions of the pH DHCR24 construct were generated and various reporter gene plasmids were transfected into HepG2 ( A ) or SK-N-MC ( B ) cells. Plasmid constructs containing different sequential portions of the DHCR24 promoter are schematized on the left and their corresponding luciferase activities are shown in the histograms on the right. The transcription factor-binding sites are identified. Cholesterol availability was modified by the incubation of cells with lovastatin (Lov, 10 μM) or LDL (30 μg of cholesterol/ml) for 24 h. Transcriptional activation was measured by the dual-luciferase reporter assay. Bars indicate the relative firefly luciferase activity normalized to Renilla luciferase activity used as the internal control of transfection efficiency. Results are the means±S.E.M. for three independent experiments.

Article Snippet: The promoter region of the human DHCR24 gene was generated by PCR amplification using HepG2 cell genomic DNA as a template and Platinum® Pfx DNA Polymerase (Invitrogen).

Techniques: Clone Assay, Plasmid Preparation, Construct, Generated, Transfection, Luciferase, Binding Assay, Modification, Incubation, Activation Assay, Reporter Assay, Activity Assay, Control

Journal: Bioscience Reports

Article Title: Promoter analysis of the DHCR24 (3β-hydroxysterol Δ 24 -reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

doi: 10.1042/BSR20120095

Figure Lengend Snippet: ( A ) Sequence of the consensus SRE, SRE in the DHCR24 promoter and in the mutated DHCR24 promoter. ( B ) Transcriptional activity assay of the wild-type and mutated SRE in the DHCR24 promoter. The wildtype or the SRE-mutated vectors were transfected into HepG2 cells. Cholesterol availability was modified by the incubation of cells with lovastatin (Lov, 10 μM) or LDL (30 μg of cholesterol/ml) for 24 h, and transcriptional activation was measured by the dual-luciferase reporter assay. Bars indicate the relative firefly luciferase activity normalized to Renilla luciferase activity used as an internal control of transfection efficiency. Results are the means±S.E.M. for three independent experiments. *** P <0.001 against different treatment within the same plasmid; # P <0.05 and ### P <0.001 against pH DHCR24 within the same treatment by two-way ANOVA.

Article Snippet: The promoter region of the human DHCR24 gene was generated by PCR amplification using HepG2 cell genomic DNA as a template and Platinum® Pfx DNA Polymerase (Invitrogen).

Techniques: Sequencing, Activity Assay, Transfection, Modification, Incubation, Activation Assay, Luciferase, Reporter Assay, Control, Plasmid Preparation

Journal: Bioscience Reports

Article Title: Promoter analysis of the DHCR24 (3β-hydroxysterol Δ 24 -reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

doi: 10.1042/BSR20120095

Figure Lengend Snippet: HepG2 cells incubated with lovastatin (Lov, 10 μM) or LDL (30 μg of cholesterol/ml) were subjected to ChIP using an antibody against SREBP-2 or IgG as a negative control. Co-precipitated DNA was amplified by PCR using primers flanking the SRE-binding site in the DHCR24 promoter as described in the Experimental section. The specific amplification of the immunoprecipitated SRE of the LDLR promoter was used as a positive control. Results are the means±S.E.M. for four independent experiments. * P <0.05 against the control condition by the paired Student's t test.

Article Snippet: The promoter region of the human DHCR24 gene was generated by PCR amplification using HepG2 cell genomic DNA as a template and Platinum® Pfx DNA Polymerase (Invitrogen).

Techniques: Incubation, Negative Control, Amplification, Binding Assay, Immunoprecipitation, Positive Control, Control

Journal: Virology

Article Title: Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.

doi: 10.1006/viro.2002.1701

Figure Lengend Snippet: FIG. 1. Expression of HCV NS 2 sequences in human hepatoma cells. Human Huh7 hepatoma cells were transfected with different expres- sion vectors and cell lysates were prepared 48 h posttransfection for Western blots. NS2 expression was detected with rabbit polyclonal antiserum against NS2. Lane 1: pCMVLIC-NS2/amino acids (AA) 827- 1026/STOP 828; Lane 2: empty vector pCMVLIC; Lane 3: GFP transfec- tion control construct (pGreenlantern; Life Technologies; Eggenstein, Germany); Lane 4: pCMVLIC-NS2 AA 810-1026 (full-length NS2); Lane 5 pCMVLIC-NS2/AA827–1026 (N-terminal truncation).

Article Snippet: HepG2 (human hepatoma), Jurkat (human CD4 Lymphoma), and HeLa (human cervix carcinoma) cells were purchased from DSMZ (Braunschweig, Germany); Huh 7 (human hepatoma) and NIH 3T3 (murine fibroblasts) cells were obtained from ATCC (Rockville, MD).

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Control, Construct

Journal: Virology

Article Title: Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.

doi: 10.1006/viro.2002.1701

Figure Lengend Snippet: FIG. 2. 2A HCV NS2 inhibits CMV immediate early promoter. HepG2 cells were cotransfected with different HCV expression vectors and a CMV-driven luciferase reporter plasmid. Cotransfection with an HCV NS2 expression vector decreased luciferase activity by 70–80% relative to vector control, while no significant reduction was observed after cotransfection with other expression constructs encoding for HCV core, E1, or p7 protein. (B) The inhibitory effect of HCV NS2 is dose-dependent. HepG2 cells were cotransfected with constant amounts of luciferase reporter construct and increasing amounts of NS2 expression plasmid adding empty vector to keep the total amount of transfected DNA constant in all transfections. (C) The inhibitory effect of HCV NS2 in non-hepatic cell lines. Huh 7 (human hepatoma), HeLa (human cervix carcinoma), Jurkat (human lymphoma), and NIH 3T3 cells (murine fibroblasts) were cotransfected with equal amounts of CMV-driven luciferase reporter plasmid and pCMVLIC-NS2/AA810–1026 (open bars) or empty vector control (filled bars). Cotransfection with the NS2 expression vector resulted in significantly decreased reporter gene activity with some variability in cell lines of different origin. (D) HCV NS2 inhibits multiple cellular and viral promoters/enhancers. The effect of HCV NS2 luciferase reporter-gene constructs under the control of different structural promoter or enhancer elements was assessed in HepG2 cells. HCV NS2 sequences inhibited reporter-gene expression to varying extents from human ferrochelatase promoter, an NFkappa B-luciferase construct, a full-length and a minimal human TNF alpha promoter without NFkappa B binding sites, and two viral promoters (SV-40 and CMV immediate early promoter). All data in this figure are given as mean standard deviation of luciferase activity relative to vector control determined 48 h after transfection.

Article Snippet: HepG2 (human hepatoma), Jurkat (human CD4 Lymphoma), and HeLa (human cervix carcinoma) cells were purchased from DSMZ (Braunschweig, Germany); Huh 7 (human hepatoma) and NIH 3T3 (murine fibroblasts) cells were obtained from ATCC (Rockville, MD).

Techniques: Expressing, Luciferase, Plasmid Preparation, Cotransfection, Activity Assay, Control, Construct, Transfection, Gene Expression, Binding Assay, Standard Deviation

Journal: Virology

Article Title: Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.

doi: 10.1006/viro.2002.1701

Figure Lengend Snippet: FIG. 4. Transient expression of HCV NS2 is not cytotoxic. HepG2 or HeLa tet-off cells were transiently transfected with pCMVLIC-Core, pCMVLIC NS2/AA 810-1026, and vector control (HepG2) or pBiEGFP- Core, pBiEGFP-NS2/AA 810-1026, or pBiEGFP-NS2 antisense control (HeLa tet-off) and expression from tetracycline-responsive promoter was induced as described above. The regular 10% FCS medium was replaced by 2% FCS medium 16 h after transfection and LDH activity was determined from the supernatant 48 h after transfection using a commercially available cytotoxicity detection kit (Roche Molecular Di- agnostics, Mannheim, Germany).

Article Snippet: HepG2 (human hepatoma), Jurkat (human CD4 Lymphoma), and HeLa (human cervix carcinoma) cells were purchased from DSMZ (Braunschweig, Germany); Huh 7 (human hepatoma) and NIH 3T3 (murine fibroblasts) cells were obtained from ATCC (Rockville, MD).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Activity Assay

Journal: Virology

Article Title: Hepatitis C virus NS2 protein inhibits gene expression from different cellular and viral promoters in hepatic and nonhepatic cell lines.

doi: 10.1006/viro.2002.1701

Figure Lengend Snippet: FIG. 6. The amino-terminal portion of HCV NS2 mediates the inhib- itory effect. Expression vectors were constructed encompassing differ- ent parts of HCV NS2 and adjacent regions in pCMVLIC. Cotransfection of HepG2 cells was carried out as described for Fig. 2A. The inhibitory effect on luciferase activity was observed only after cotransfection with sequences which included the amino-terminal portion of NS2 (amino acids 810-1026, 810–940, 730-1006).

Article Snippet: HepG2 (human hepatoma), Jurkat (human CD4 Lymphoma), and HeLa (human cervix carcinoma) cells were purchased from DSMZ (Braunschweig, Germany); Huh 7 (human hepatoma) and NIH 3T3 (murine fibroblasts) cells were obtained from ATCC (Rockville, MD).

Techniques: Inhibition, Expressing, Construct, Cotransfection, Luciferase, Activity Assay

Journal: Frontiers in pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo .

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: FIGURE 1 | EEAC reduces viability and induces apoptosis in human HCC cells. (A) Effects of EEAC on cell viability in HpeG2 and SMMC-7721 cells. Cells were treated with various concentrations of EEAC for 24 and 48 h, respectively. Cell viability was assessed by MTT assays. (B) Effects of EEAC on apoptosis in HCC cells. Cells were exposed to EEAC at indicated concentrations for 48 h and percentage of apoptotic cells was determined by flow cytometry after Annexin V/PI double staining. Data were shown as mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. control. Protein levels of PARP and cleaved-PARP in HCC cells were explored by western blotting following EEAC treatment for 24 h. (C) Western blot analyses for protein levels of cleaved-caspase-9, -8, and -3 in HCC cells. Results shown are representative of three independent experiments.

Article Snippet: Human HCC cells (PLC/PRF/5 and HepG2) and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, United States).

Techniques: Cytometry, Double Staining, Control, Western Blot

Journal: Frontiers in pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo .

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: FIGURE 2 | EEAC dampens HCC cell migration and invasion. (A) migration assays and (B) invasion assays were performed in HepG2 and SMMC-7721 cells for 48 h after pre-treatment with indicated concentrations of EEAC for 24 h. Representative images are shown on the left and the quantification of 4 randomly selected fields was shown on the right. Scale bar = 100 µm. Data were shown as mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. control.

Article Snippet: Human HCC cells (PLC/PRF/5 and HepG2) and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, United States).

Techniques: Migration, Control

Journal: Frontiers in pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo .

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: FIGURE 3 | EEAC inhibits activation and lowers mRNA level of STAT3 in human HCC cells. Cells were treated with indicated concentrations of EEAC for 24 h, and then protein levels of (A) p-STAT3 and STAT3 and (B) p-JAK2 and JAK2 were determined by Western blot analyses. The relative protein levels were analyzed by Image J software. (C) mRNA levels of STAT3 (upper panel) and JAK2 (lower panel) in HepG2 and SMMC-7721 cells were detected by qRT-PCR. Data were shown as mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. the corresponding control.

Article Snippet: Human HCC cells (PLC/PRF/5 and HepG2) and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, United States).

Techniques: Activation Assay, Western Blot, Software, Quantitative RT-PCR, Control

Journal: Frontiers in pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo .

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: FIGURE 4 | EEAC reduces STAT3 nuclear pool and suppresses STAT3-luciferase reporter activity in human HCC cells. (A) Protein levels of STAT3 in cytoplasmic and nuclear extracts. HepG2 cells were treated with indicated concentrations of EEAC for 24 h. Protein levels of STAT3 were determined by Western blot analyses (left) and relative expression levels were analyzed by Image J software (right). GAPDH and SP1 were served as loading controls of cytoplasmic and nuclear extractions, respectively. (B) HepG2 cells were transfected with STAT3-luciferase reporter plasmid with a PRL-CMV construct for 48 h, and then treated with EEAC at indicated concentrations for another 24 h. Transcriptional activity of STAT3 was measured by the dual-luciferase reporter assay. Data were shown as mean ± SD of three independent experiments. ∗∗P < 0.01 vs. the control.

Article Snippet: Human HCC cells (PLC/PRF/5 and HepG2) and normal human liver-derived cells (MIHA) were purchased from American Type Culture Collection (ATCC, United States).

Techniques: Luciferase, Activity Assay, Western Blot, Expressing, Software, Transfection, Plasmid Preparation, Construct, Reporter Assay, Control