Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Cell viability ( a ) and DNA biosynthesis ( b ) in MCF7 cells treated for 24 h with the following concentrations of drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. The mean values ± standard error (SEM) from the 3 experiments, performed in duplicates, are presented at p < 0.05.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Cytometric assay of apoptosis in MCF7 cells. After 24 h of incubation with selected drugs (0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM of troglitazone), the cells were stained with fluorescent dyes using Solution 5 (ChemoMetec ® ) containing Propidium Iodide and VB48. Cells in the upper polygon are apoptotic or dead. Cells in the lower right polygon are healthy living cells.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Immunofluorescence staning of p53 expression and its translocation to the nuceli in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM of troglitazone. Blue indicates the nuceli, and the red fluorescence represents p53 expression.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Immunofluorescence, Expressing, Translocation Assay, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Western blot analysis of COX2 expression in MCF7 cells after 24 h of incubation, with 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM of troglitazone. For the experiment, 40 µg/lane of protein from the lysates was used. The expression of Β-actin serves as a loading control.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Western Blot, Expressing, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Immunofluorescence staining of PPARγ and its translocation to the nuclei in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib, and 0.02 mM troglitazone. Blue indicates the nuceli, and the red fluorescence represents PPARγ localization and expression.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Immunofluorescence, Staining, Translocation Assay, Fluorescence, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Western blot analysis for PPARδ expression in MCF7 cells incubated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. For the experiment, 40 µg/lane of protein from the lysates was used.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Western Blot, Expressing, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Western blot for PRODH/POX expression in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. For the experiment, 40 µg/lane of protein lysates was used.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Immunofluorescence staining of PRODH/POX expression in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. Blue stainng indicates the nuceli, and red staining represents PRODH/POX expression.The inhibition of DNA biosynthesis by the studied drugs was accompanied by the inhibition of protein biosynthesis. The biosynthesis of collagen, the most abundant protein in mammalian cells, can represent the total protein synthesis. As is presented in a, NSAIDs and troglitazone provoke strong inhibitory effects on collagen biosynthesis in MCF7 cells. In the case of nonselective COX2 inhibitors, such as ibuprofen, indomethacin, diclofenac, and sulindac, the process was inhibited to 32, 28, 21, and 25% of the control, respectively. The selective COX2 inhibitor, celecoxib, and the PPARγ agonist, troglitazone, decreased the collagen biosynthesis to 27% and 19% of the control, respectively.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Immunofluorescence, Staining, Expressing, Inhibition, Control

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Collagen biosynthesis ( a ) and prolidase activity ( b ) in MCF7 cells treated for 24 h with the following concentrations of drugs: 750 µM ibuprofen; 500 µM indomethacin; 375 µM diclofenac; 60 µM sulindac; 20 µM celecoxib, and 20 µM troglitazone. The mean values ± standard error (SEM) from the 3 experiments, performed in duplicates, are presented at p < 0.005.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Fluorescence analysis of reactive oxygen species generation due to the POX activation using 0.5 µM of 2′,7′-dichlorofluorescin diacetate staining. Analysis performed on live cells using a survival chamber: 37 °C in 5% CO 2 . MCF7 cells were incubated with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone, for the 4 h. Red fluorescence intensity represents amount of generated ROS.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Fluorescence, Activation Assay, Staining, Incubation, Generated

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Fluorescence analysis of ROS generation in MCF7 in the presence of THFA (PRODH/POX inhibitor). NSAID POX activation was inhibited by 0.5 h preincubation, with 0.1 mM of THFA. Fluorescence was determined by using 0.5 µM of 2′,7′-dichlorofluorescin diacetate staining. Analysis was performed on live cells using a survival chamber: 37 °C in 5% CO 2 . After preincubation with THFA, the MCF7 cells were incubated with the following drugs: 0.75 mM ibuprofen; 0.5mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone, for the 4 h. Red fluorescence intensity represents amount of generated ROS.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Fluorescence, Activation Assay, Staining, Incubation, Generated

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Western blot for PYCR1 and PYCRL ( a ), and mTOR and p-AMPK alpha ( b ) expressions in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. For the experiment, 40 µg/lane of protein lysates was used.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis

doi: 10.3390/ijms23031510

Figure Lengend Snippet: Western blot for caspase-8, caspase-9, and caspase-7 (total and cleaved), PARP, total and cleaved, in MCF7 cells treated for 24 h with the following drugs: 0.75 mM ibuprofen; 0.5 mM indomethacin; 0.375 mM diclofenac; 0.06 mM sulindac; 0.02 mM celecoxib; and 0.02 mM troglitazone. For the experiment, 40 µg/lane of protein lysates was used. Protein expression was normalized versus β-actin expression.

Article Snippet: The celecoxib and troglitazone were products of Targetmol.

Techniques: Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: 25-Hydroxycholesterol Inhibits Adipogenic Differentiation of C3H10T1/2 Pluripotent Stromal Cells

doi: 10.3390/ijms21020412

Figure Lengend Snippet: Representative image of C3H10T1/2 mouse cells treated with a control, DMITro (500 nM DEX, 0.5 mM IBMX, 20 µg/mL Insulin and 10 µM Troglitazone), DMITro + 10 µM 25-HC, 20S, 22S or 22R hydroxycholesterols for six days, followed by oil red O staining and scanning of plates for lipid accumulation comparisons. ( A ) oil red O stanning results ( B ) The mean gray value (in pixel) of oil red O staining. The results show the average values of three replicates ( n = 3) and the SE of the means. Bars with the different letters are significantly different ( p < 0.001).

Article Snippet: Troglitazone was purchased from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: 25-Hydroxycholesterol Inhibits Adipogenic Differentiation of C3H10T1/2 Pluripotent Stromal Cells

doi: 10.3390/ijms21020412

Figure Lengend Snippet: The effects of 25-HC, 20S, 22S and 22R on Pparγ ( A ), C/ebpα ( B ), Fabp4 ( C ) and Lpl ( D ) mRNA expression induced by DMITro. Cells were treated with a control, DMITro (500 nM DEX, 0.5 mM IBMX, 20 µg/mL Insulin and 10 µM Troglitazone) and DMITro + 10 µM 20S, 25-HC, 22R or 22S for 6 days. The results show the average values of three replicates ( n = 3) and the SD of the means. Bars with the same letter are not significantly different.

Article Snippet: Troglitazone was purchased from Tocris Bioscience (Ellisville, MO, USA).

Techniques: Expressing, Control

Journal: Cell reports

Article Title: Tonic type 2 immunity is a critical tissue checkpoint controlling autoimmunity in the skin.

doi: 10.1016/j.celrep.2024.114364

Figure Lengend Snippet: Figure 4. Activation of LXR or PPARg restores fatty acid metabolism and alleviates skin inflammation in ongoing psoriasis (A and B) Psoriasiform dermatitis was induced in mice. Thickness change (%) was calculated by setting the thickness of the start date as 100% (dotted line). Representative histological images of skin sections are shown. UTX, untreated; IMQ, imiquimod. Scale bar represents 300 mm. (C–E) Results of transcriptomic analysis of the ear skin samples. (C) Enrichment scores for FA metabolism and Th17 response signature. (D) Enrichment scores for 13 representative psoriatic gene signatures (column) in the indicated group comparisons (row). The NES and p values were obtained by GSEA. *p < 0.05, **p < 0.01. (E) Representative biological processes (GO terms and clusters) altered by GW3965 or troglitazone treatment. Common (left), GW3965-specific (middle), and troglitazone-specific (right) alterations are shown. The GO pairs with high overlap of constituent genes are connected by a gray line. (F and G) Relative abundance of indicated (F) lipid classes and (G) subclasses. Data shown indicate mean ± SEM. Data represent at least two independent experiments with n = 3–5 mice per group. Each dot represents an individual mouse (C) or average of 5 mice in group (A, B, F, and G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.

Article Snippet: Biological samples Healthy human skin tissue Yonsei University College of Medicine, Severance Hospital N/A Chemicals, peptides, and recombinant proteins RPMI-1640 medium Sigma-Aldrich Cat#R8758 DMEM medium Sigma-Aldrich Cat#D6429 Dulbecco’s Phosphate-Buffered Saline (DPBS) GenDEPOT Cat#CA008 FBS Sigma-Aldrich Cat#TMS-013 Pennicillin-Streptomycin (100X) GibcoTM Cat#15140122 L-Glutamine (200 mM) GibcoTM Cat#25030081 2-Mercaptoethanol (1,000X) GibcoTM Cat#21985023 Gentamicin (50 mg/mL) GibcoTM Cat#15750078 PMA Sigma-Aldrich Cat#P1585 Ionomycin calcium salt from Streptomyces conglobatus Sigma-Aldrich Cat#I0634 Brefeldin A Solution (1000X) InvitrogenTM Cat#00-4506-51 Monensin Solution (1000X) InvitrogenTM Cat#00-4505-51 Aldara, 5% cream iNova Pharmaceuticals N/A HBSS, calcium, magnesium, no phenol red GibcoTM Cat#14025092 Collagenase IV Sigma-Aldrich Cat#C5138 DNase I Roche Cat#04536282001 Brilliant Stain Buffer BD Biosciences Cat#566349 RNAlaterTM Stabilization Solution InvitrogenTM Cat#AM7020 TRIzolTM Reagent InvitrogenTM Cat#15596018 Chloroform Junsei Cat#28560-0350 Iso-Propyl Alcohol 99.5% Reagents DUKSAN Cat#858 RIPA Cell Lysis Buffer (1X) with EDTA (SNU) GenDEPOT Cat#R4100 RIPA Cell Lysis and Extraction Buffer (MIMR) ThermoFisher Cat#89900 Xpert Protease Inhibitor Cocktail Solution (100X) (SNU) GenDEPOT Cat#P3100 HaltTM Protease Inhibitor Cocktail (100X) (MIMR) ThermoFisher Cat#78430 Ethanol Supelco Cat#1.00983 Dimethyl Sulfoxide (DMSO) VWR Cat#V0231 GW 3965 hydrochloride Tocris Cat#2474 Troglitazone Tocris Cat#3114 Troglitazone MCE Cat#HY-50935 GW9662 MCE Cat#HY-16578 AS1517499 Selleckchem Cat#S8685 AS1517499 MCE Cat#HY-100614 Mouse recombinant IL-1b Peprotech Cat#211-11B Mouse IL-23 Recombinant Protein eBioscienceTM Cat#14-8231-63 Recombinant Human IL-17A Peprotech Cat#200-17 Recombinant Human IL-22 Peprotech Cat#200-22 Recombinant Human TNF-a Peprotech Cat#300-01A Recombinant Human IL-2 R&D Cat#202-IL Recombinant Human IL-1b BioLegend Cat#579404 Recombinant Human IL-23 Peprotech Cat#200-23 (Continued on next page) Cell Reports 43, 114364, July 23, 2024 17

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Characterization of a long-term mouse primary liver 3D tissue model recapitulating innate-immune responses and drug-induced liver toxicity

doi: 10.1371/journal.pone.0235745

Figure Lengend Snippet: a Coculture spheroids were treated for 48 h with 5 μM and 25 μM Cyclosporine A (CsA) and stained with DAPI (blue) and for neutral lipids (green) using immunofluorescence. Scale bar = 50 μm b Quantitative analysis of neutral-lipid accumulation in CsA-treated spheroids. Data are represented as accumulation in % with respect to vehicle control (ctrl) ± standard deviation. (n = 8 spheroids per condition). c Coculture spheroids were treated with different concentrations of Cyclophosphamide (CPZ) and Troglitazone for 24 h and analyzed for BSEP gene expression via qRT-PCR. Data are expressed as x-fold change compared to vehicle control (ctrl) and corrected to GAPDH mRNA ± standard deviation (n = 4). Statistical significance is indicated * (p-value ≤ 0.05; Student’s t-test), ** (p-value ≤ 0.01; Student’s t-test), and *** (p-value ≤ 0.001; Student’s t-test), ns meant not significant (p-value > 0.05).

Article Snippet: On day 7 post seeding, coculture spheroids were treated with different concentrations of Chlorpromazine (CPZ, Sigma-Aldrich, Switzerland) and Troglitazone (LKT Labs, USA) for 24 h. Bile salt export pump (BSEP) downregulation was measured from pooled spheroids via qRT-PCR.

Techniques: Staining, Immunofluorescence, Control, Standard Deviation, Gene Expression, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

doi: 10.3389/fmicb.2018.03257

Figure Lengend Snippet: Troglitazone inhibits HBV infection. (A) Chemical structure of troglitazone. (B) Schematic representation of the protocol for treating HepG2-hNTCP-C4 cells with compounds and HBV. HepG2-hNTCP-C4 cells were pretreated with compounds for 2 h and then were inoculated with HBV in the presence or absence of compounds for 16 h. After the washing out of free HBV and compounds, the cells were cultured in the absence of compounds for an additional 12 days, and HBV infection was evaluated by quantifying each of the markers shown in (C–G) . Black and dotted boxes indicate the periods with and without treatment, respectively. (C–H) HepG2-hNTCP-C4 cells treated with or without 100 nM preS1 peptide or 25 μM troglitazone were inoculated with HBV according to the protocol shown in (B) . HBV infection was examined by ELISA detection of HBs (C) and HBe (D) antigens in the culture supernatant, by real-time PCR detection of HBV DNA (E) and cccDNA (F) in the cells, and by immunofluorescence analyses to detect HBc antigen (G) in the cells. Cell viability was also measured by the MTT assay (H) . Red and blue signals in (G) indicate HBc antigen and the nucleus, respectively. Data are shown as mean ± SD. Statistical significance was determined using a two-tailed non-paired Student's t -test ( ** P < 0.01).

Article Snippet: Troglitazone was purchased from AdooQ BioScience.

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, MTT Assay, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

doi: 10.3389/fmicb.2018.03257

Figure Lengend Snippet: Analysis of troglitazone analogs. (A) Chemical structures of troglitazone analogs. The structures shared in common among troglitazone and its analogs are shown by red and blue. (B) HepG2-hNTCP-C4 cells were inoculated with HBV following treatment with 100 nM preS1 peptide or 25 μM troglitazone analogs as shown in Figure . HBV infection was evaluated by quantifying HBs antigen in the culture supernatant. (C,D) Primary human hepatocytes were treated with or without the indicated compounds (100 nM preS1 peptide and 3, 10, and 30 μM of troglitazone or ciglitazone) and inoculated with HBV as shown in Figure . HBs antigen (C) secreted into the culture supernatant was detected by ELISA. Cell viability was also measured by the MTT assay (D) . Data are shown as mean ± SD. Statistical significance was determined using a two-tailed non-paired Student's t -test ( * P < 0.05, ** P < 0.01).

Article Snippet: Troglitazone was purchased from AdooQ BioScience.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MTT Assay, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

doi: 10.3389/fmicb.2018.03257

Figure Lengend Snippet: The anti-HBV activity of troglitazone is not mediated via PPARγ activity. (A–C) HepG2-hNTCP-C4 cells were transfected with a reporter plasmid carrying binding elements for PPAR (PPRE) together with expression plasmids encoding PPARγ and RXRα (A) . The cells were treated with or without 25 μM troglitazone, 25 μM pioglitazone (B) , or 25 μM troglitazone in the presence or absence of 1 mM SR-202, a PPARγ antagonist, for 24 h (C) , and luciferase reporter activity was measured. (D,E) HepG2-hNTCP-C4 cells treated with or without 25 μM troglitazone, 25 μM pioglitazone (D) , or 25 μM troglitazone in the presence or absence of 1 mM SR-202 (E) were subjected to the HBV infection assay according to the scheme shown in Figure . HBV infection was evaluated by quantifying HBs antigen in the culture supernatant. Data are shown as mean ± SD. Statistical significance was determined using a two-tailed non-paired Student's t -test (N.S.; not significant, ** P < 0.01).

Article Snippet: Troglitazone was purchased from AdooQ BioScience.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Binding Assay, Expressing, Luciferase, Infection, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

doi: 10.3389/fmicb.2018.03257

Figure Lengend Snippet: Troglitazone inhibits HBV internalization. (A) Schematic representation of the HBV life cycle. The blue boxes show the life cycle processes that were examined in the experiments in (B,C,E,F) . (B) HBV replication was evaluated by quantifying HBV DNA in HepG2.2.15.7 cells treated for 6 days with or without 1 μM entecavir (as a positive control) or 25 μM troglitazone. (C) HDV infection assay. HepG2-hNTCP-C4 cells were inoculated with HDV in the presence or absence of 100 nM preS1 peptide or 25 μM troglitazone, according to the protocol shown in Materials and Methods. HDV infection was evaluated by quantifying HDV RNA in the cells by real-time RT-PCR. (D) HCV infection was evaluated using the HCV pseudoparticle system, as described in Materials and Methods, following treatment with or without 10 nM bafilomycin A1 as a positive control or 25 μM troglitazone. (E) HBV preS1-mediated attachment to host cells was examined in HepG2-hNTCP-C4 cells exposed for 30 min at 37°C to TAMRA-labeled preS1 peptide (preS1 probe) in the presence or absence of 100 nM non-labeled preS1 peptide or 25 μM troglitazone. Red and blue signals indicate preS1 probe and the nucleus, respectively. ( Fa ) Schematic of the experimental protocol used for examining HBV internalization. HepG2-hNTCP-C4 cells were treated with HBV at 4°C for 3 h to allow HBV-cell attachment without internalization into the cells. After free HBV was washed out, the cells were cultured for 16 h at 37°C in the presence or absence of 25 μM troglitazone to allow viral internalization. The cells then were trypsinized and extensively washed to remove the cell surface HBV, and HBV DNA in the cells was quantified (b) . (c) Schematic of the experimental protocol used for examining preS1 internalization. HepG2-hNTCP-C4 cells were exposed for 8 h at 37°C to preS1 probe (red) in the presence or absence of 25 μM troglitazone or pioglitazone, and then were stained for NTCP (green) and the nucleus (blue) by immunofluorescence analysis (d) . Merged patterns are shown in the bottom panels. Data are shown as mean ± SD. Statistical significance was determined using a two-tailed non-paired Student's t -test ( ** P < 0.01).

Article Snippet: Troglitazone was purchased from AdooQ BioScience.

Techniques: Positive Control, Infection, Quantitative RT-PCR, Labeling, Cell Attachment Assay, Cell Culture, Staining, Immunofluorescence, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

doi: 10.3389/fmicb.2018.03257

Figure Lengend Snippet: Inhibition of NTCP oligomerization by peptides or troglitazone impedes HBV preS1-NTCP internalization. ( Aa,b ) Effect of NTCP peptides (aa 1–20, aa 221–240, and aa 271–290) on preS1 internalization as described in Figures . Merged patterns of green (NTCP), red (preS1 probe), and blue (nucleus) signals are shown. Intracellular dotted yellow signals (the internalized preS1-NTCP) were clearly observed in the control and with aa 1–20, while those signals were decreased with aa 221–240 and even more strongly decreased with aa 271–290. (c) HBV infection of HepG2-hNTCP-C4 cells was examined as described in Figure following treatment with or without the indicated peptides [preS1 or NTCP peptides (aa 1–20, aa 221–240, or aa 271–290)] using detection of HBs antigen at 12 days post-infection. (B) Effect of troglitazone on NTCP oligomerization was evaluated by PLA, as shown in Figure . PLA signal was detected in HepG2 cells overexpressing both myc-NTCP and HA-NTCP following treatment with or without preS1 probe in the presence or absence of troglitazone or pioglitazone. (a) The lower panels show the PLA signal (green) only, and the upper panels are the merged images of green (PLA signal), red (preS1 probe), and blue (nucleus). (b) PLA signals in the experiment in (a) were quantified using Dynamic Cell Count (KEYENCE). Signal was significantly reduced by troglitazone, but not by pioglitazone. Data are shown as mean ± SD. Statistical significance was determined using a two-tailed non-paired Student's t -test (N.S.; not significant, * P < 0.05, ** P < 0.01).

Article Snippet: Troglitazone was purchased from AdooQ BioScience.

Techniques: Inhibition, Control, Infection, Cell Counting, Two Tailed Test