Journal: bioRxiv

Article Title: Development of in vitro test methods in a model of human pancreatic β-cells to identify metabolism disrupting chemicals with diabetogenic activity

doi: 10.1101/2022.03.22.485271

Figure Lengend Snippet: EndoC-βH1 (A) and INS-1E (B) cells were treated with vehicle (DMSO) or BPA (10 pM or 1 nM) in the absence or presence of 1 μM ICI 182,780 for 24 h. EndoC-βH1 (C) and INS-1E (D) cells were treated with vehicle (DMSO) or TBT (20 nM or 200 nM) in the absence or presence of 100 nM T0070907 for 24 h. Apoptosis was evaluated using HO and PI staining. Data are shown as means ± SEM of four independent experiments. ***p≤0.001 vs its respective Vehicle; #p≤0.05, ##p≤0.01, and ###p≤0.001 as indicated by bars. Two-way ANOVA.

Article Snippet: ICI 182,780 (Cat. No. 1047) and T0070907 (Cat. No. HY-13202) were obtained from Tocris Cookson Ltd (Avonmouth, UK) and MedChem Express (New Jersey, USA), respectively.

Techniques: Staining

Journal: PLoS ONE

Article Title: Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells

doi: 10.1371/journal.pone.0104397

Figure Lengend Snippet: Representative Western blot analysis of PPAR-γ in protein extracts from HSCs and ISEMFs treated with plasma and incubated for 4 hrs. As indicated, cells were pre-treated with NAC. Similar results were obtained in three independent experiments. β-actin was used as a loading control (panel A ). Cells were treated for 30 min with 50 µM PPAR-γ receptor antagonist T0070907, exposed to plasma, and then incubated under standard conditions for 24 hrs. IL-6 (panel B ) and IL-1β (panel C ) were assessed in the conditioned medium by ELISA. Data are reported as mean±SE of results collected in nine independent experiments, each performed in triplicate. * denotes P<0.05 vs non treated cells.

Article Snippet: HSCs and ISEMFs were incubated for 30 min with PPAR-γ receptor antagonist T0070907 (50 μM, Tocris, UK) or with vehicle alone.

Techniques: Western Blot, Clinical Proteomics, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: Retinoid X Receptor Activation During Adipogenesis of Female Mesenchymal Stem Cells Programs a Dysfunctional Adipocyte

doi: 10.1210/en.2018-00056

Figure Lengend Snippet: Chemical antagonists reveal PPARγ- and RXR-dependent differences in ROSI- vs rexinoid-differentiated MSCs. (A–C) MSCs were differentiated into adipocytes in the presence of MDI and ligands (500 nM ROSI, 100 nM 4204, 50 nM TBT, 0.1% DMSO). On day 7 of differentiation, chemical antagonists of PPARγ (T0070907, 1 μM) or RXR (HX531, 10 μM) were added to the culture medium through the end of differentiation (see Supplemental Fig. 6A). RNA was collected and analyzed by qPCR for gene expression of (A) classic PPARγ and RXR targets, (B) IFN genes, or (C) BAT markers. A two-way ANOVA and a Tukey multiple comparison within each treatment group were used: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (D and E) MSCs were differentiated for 7 d in the presence of MDI and ligands as in (A)–(C). On day 7, mouse recombinant IFNα (1000 U/mL) was added to ROSI samples and the JAK inhibitor TCN (5 μM) was added to 4204 and TBT samples (see Supplemental Fig. 6B). Gene expression of (D) IFN genes and (E) BAT markers was analyzed by qPCR. By an unpaired t test: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. All data are represented as the mean ± SEM.

Article Snippet: Chemicals All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated: dimethyl sulfoxide (Thermo Fisher Scientific, Waltham, MA), TBT, ROSI (Cayman Chemical, Ann Arbor, MI), 4204 (also known as AGN194204; a gift from Rosh Chandraratna, IO Therapeutics, Santa Ana, CA), T0070907 (Enzo Life Sciences, Farmingdale, NY), HX531 (a gift from Claes Bavik, Acucela, Inc., Seattle, WA), dexamethasone, human recombinant insulin, isobutylmethylxanthine, triiodothyronine, mouse recombinant interferon (IFN) α (PBL Assay Science, Piscataway, NJ), tofacitinib (TCN; Selleck Chemicals, Houston, TX), 2-deoxy- d -glucose, 2-deoxy- d -[ 3 H]-glucose (American Radiolabeled Chemicals, St. Louis, MO), Nile Red, Hoechst 33342, and collagenase D. Mesenchymal stem cell culture and differentiation Mouse bone marrow–derived MSCs (OriCell, Cyagen Biosciences, Santa Clara, CA) from female C57BL/6 mice were stored at passage 9 in liquid N 2 .

Techniques: Gene Expression, Comparison, Recombinant

Journal: Biological & pharmaceutical bulletin

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

doi: 10.1248/bpb.b13-00024

Figure Lengend Snippet: Fig. 1. The Effects of T0070907 and α-Tocopherol on Adipogenesis in Maturing 3T3-L1 Adipocytes

Article Snippet: Materials The reagents used in the study were purchased as follows: 3,3,5-triiode-L-thyronine (T3), L-thyroxine (T4), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, α-tocopherol, α-tocotrienol, butylhydroxyanisole, astaxanthin, Hoechst33342, GW1929, T0070907, SR-202 and Oil Red O from Sigma Aldrich (St. Louis, MO, U.S.A.), tertbutylhydroquinone and 10% formalin solution from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), γ-tocotrienol from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceutical (Tokyo, Japan), d-biotin from Nacalai Tesque (Kyoto, Japan), Dual-Luciferase Reporter Assay System from Promega (Osaka, Japan), Fugene HD from Roche Diagnostics Japan (Tokyo, Japan).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

doi: 10.1248/bpb.b13-00024

Figure Lengend Snippet: Fig. 2. T0070907 Induced Apoptosis in Early-Differentiated Immature 3T3-L1 Adipocytes

Article Snippet: Materials The reagents used in the study were purchased as follows: 3,3,5-triiode-L-thyronine (T3), L-thyroxine (T4), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, α-tocopherol, α-tocotrienol, butylhydroxyanisole, astaxanthin, Hoechst33342, GW1929, T0070907, SR-202 and Oil Red O from Sigma Aldrich (St. Louis, MO, U.S.A.), tertbutylhydroquinone and 10% formalin solution from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), γ-tocotrienol from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceutical (Tokyo, Japan), d-biotin from Nacalai Tesque (Kyoto, Japan), Dual-Luciferase Reporter Assay System from Promega (Osaka, Japan), Fugene HD from Roche Diagnostics Japan (Tokyo, Japan).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

doi: 10.1248/bpb.b13-00024

Figure Lengend Snippet: Fig. 3. T0070907 Induces Cytotoxicity in an Immature Adipocyte- Specific Manner

Article Snippet: Materials The reagents used in the study were purchased as follows: 3,3,5-triiode-L-thyronine (T3), L-thyroxine (T4), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, α-tocopherol, α-tocotrienol, butylhydroxyanisole, astaxanthin, Hoechst33342, GW1929, T0070907, SR-202 and Oil Red O from Sigma Aldrich (St. Louis, MO, U.S.A.), tertbutylhydroquinone and 10% formalin solution from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), γ-tocotrienol from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceutical (Tokyo, Japan), d-biotin from Nacalai Tesque (Kyoto, Japan), Dual-Luciferase Reporter Assay System from Promega (Osaka, Japan), Fugene HD from Roche Diagnostics Japan (Tokyo, Japan).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

doi: 10.1248/bpb.b13-00024

Figure Lengend Snippet: Fig. 5. Adipocyte-Specific and PPARγ-Independent Cytotoxicity of T0070907

Article Snippet: Materials The reagents used in the study were purchased as follows: 3,3,5-triiode-L-thyronine (T3), L-thyroxine (T4), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, α-tocopherol, α-tocotrienol, butylhydroxyanisole, astaxanthin, Hoechst33342, GW1929, T0070907, SR-202 and Oil Red O from Sigma Aldrich (St. Louis, MO, U.S.A.), tertbutylhydroquinone and 10% formalin solution from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), γ-tocotrienol from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceutical (Tokyo, Japan), d-biotin from Nacalai Tesque (Kyoto, Japan), Dual-Luciferase Reporter Assay System from Promega (Osaka, Japan), Fugene HD from Roche Diagnostics Japan (Tokyo, Japan).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ)-independent specific cytotoxicity against immature adipocytes induced by PPARγ antagonist T0070907.

doi: 10.1248/bpb.b13-00024

Figure Lengend Snippet: Fig. 6. Suppression of T0070907-Induced Cytotoxicity by Antioxidants

Article Snippet: Materials The reagents used in the study were purchased as follows: 3,3,5-triiode-L-thyronine (T3), L-thyroxine (T4), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, α-tocopherol, α-tocotrienol, butylhydroxyanisole, astaxanthin, Hoechst33342, GW1929, T0070907, SR-202 and Oil Red O from Sigma Aldrich (St. Louis, MO, U.S.A.), tertbutylhydroquinone and 10% formalin solution from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), γ-tocotrienol from Santa Cruz Biotechnologies (Santa Cruz, CA, U.S.A.), Dulbecco’s modified Eagle’s medium (DMEM) from Nissui Pharmaceutical (Tokyo, Japan), d-biotin from Nacalai Tesque (Kyoto, Japan), Dual-Luciferase Reporter Assay System from Promega (Osaka, Japan), Fugene HD from Roche Diagnostics Japan (Tokyo, Japan).

Techniques:

Journal: Environmental Health Perspectives

Article Title: Effects of Perinatal Exposure to Dibutyltin Chloride on Fat and Glucose Metabolism in Mice, and Molecular Mechanisms, in Vitro

doi: 10.1289/EHP3030

Figure Lengend Snippet: PPAR γ antagonist T0070907 effects on DBT-induced adipogenesis in human and mouse MSCs. Adipogenesis was induced in (A, B) human and (C, D) mouse MSCs with adipogenic cocktail (MDI) and 100 nM DBT in the presence or absence of the PPAR γ antagonist T0070907 ( 100 nM ). (A, C) Lipid accumulation is presented as the ratio of relative fluorescence units (RFUs) of Nile Red and Hoechst. (B, D) Gene expression levels of adipogenic markers expressed as fold induction over vehicle. Media with adipogenic cocktail and ligands was replaced every 3 d for 14 d. Fresh T0070907 was added every 8 h throughout the experiment. One-way analysis of variance (ANOVA) was conducted to compare DMSO and the different concentrations of DBT, followed by Dunnett’s post-hoc test. Unpaired t -test was conducted for the positive controls ROSI and TBT versus vehicle. Student’s t -test was performed to compare every treatment in the presence or absence of T0070907. All data are expressed as the average of 6 replicates ± SEM . Note: C / EBP α , CCAAT/Enhancer Binding Protein Alpha; DBT, dibutyltin; DMSO,dimethylsulfoxide; Fabp4,fatty acid binding protein-4; Fsp27, fat-specific protein-27; LPL, lipoprotein lipase; h/mMSCs, human/mouse mesenchymal stem cells; PPAR γ 2 , peroxisome proliferator–activated receptor gamma; ROSI, rosiglitazone; SEM, standard error of the mean; TBT, tributyltin. * p ≤ 0.05 in comparison with vehicle (DMSO). # p ≤ 0.05 comparing T0070907 samples with DMSO samples within the same treatment.

Article Snippet: Rosiglitazone (ROSI) was purchased from Cayman Chemicals, and T0070907 from Enzo Life Sciences.

Techniques: Fluorescence, Expressing, Binding Assay

Journal: Scientific Reports

Article Title: Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

doi: 10.1038/srep42104

Figure Lengend Snippet: ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) at various times after initiation of differentiation. Percent raw triglyceride inhibition of half maximal rosiglitazone per well for T0070907 at 7 days ( A ), 10 days ( B ), and 14 days ( C ). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for T0070907 at 7 days ( D ), 10 days ( E ), and 14 days ( F ). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for T0070907 at 7 days ( G ), 10 days ( H ), and 14 days ( I ). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p < 0.05, as per linear mixed model in SAS 9.4.

Article Snippet: Lastly, T0070907 and GW9662 exhibited more potent inhibition of triglyceride accumulation via PPARγ antagonism in Zenbio 3T3-L1 and OP9 cells, though decreased DNA content was only observed in the OP9 cells.

Techniques: Staining, Inhibition, Control, Concentration Assay

Journal: Scientific Reports

Article Title: Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

doi: 10.1038/srep42104

Figure Lengend Snippet: Comparison of Adipogenic Activity by Test Compounds.

Article Snippet: Lastly, T0070907 and GW9662 exhibited more potent inhibition of triglyceride accumulation via PPARγ antagonism in Zenbio 3T3-L1 and OP9 cells, though decreased DNA content was only observed in the OP9 cells.

Techniques: Comparison, Activity Assay

Journal: Life Science Alliance

Article Title: Connexin 32 constrains a mesenchymal-like switch in differentiated urothelium and luminal cancers

doi: 10.26508/lsa.202503427

Figure Lengend Snippet: Gene expression changes by normal human urothelial cells after in vitro differentiation and analysis of RNAseq. (A, B) panel of archetypical urothelial differentiation-restricted genes for validation purposes and (B) connexin genes. The RNAseq data series represents three independent NHU cell lines differentiated in vitro using either ABSCa or TZPD protocols, for 7 and 6 d, respectively ( ; ; ). Differentiation was confirmed from up-regulation of transcripts for the uroplakins ( UPK1A , UPK1B , UPK2 , UPK3A , and UPK3B ) , tight junction-associated claudins ( CLDN3 , CLDN4 , and CLDN7 ) , zonula occludens ( TJP2 and TJP3 ) , GATA3 , and transitional cytokeratins KRT13 and KRT20 as well as PPARG . Reciprocal loss was seen of squamous-associated KRT14 and TP63 . Data are represented as Log 2 fold-change TPM (transcripts per million) values, from differentiated NHU cell cultures relative to donor-matched undifferentiated parallel cultures. Error bars ± SD. Dotted line at ±1 indicates significant twofold change. (C) Cx32 immunofluorescence labelling of NHU cells cultured on glass slides for 6 d in vehicle control (DMSO) and TZPD differentiated, or undifferentiated and ABSCa differentiated, conditions. Nuclei were counterstained (blue) with Hoechst 33258. Scale bar = 25 μm. Red and green immunolabelling are the consequence of using secondary antibodies conjugated to different fluorochromes. (D) Left panel—Cx32 immunoblotting of NHU cells cultured for 6 d in 0.1% DMSO (vehicle control—Ctrl), 1 μM TZ, 1 μM PD153035, TZPD combined treatment, or TZPD with 5 μM T0070907. Right panel—Cx32 immunoblotting of NHU cells cultured in undifferentiated (Ctrl), ABSCa differentiated, or ABSCa with 5 μM T0070907. Monomeric and dimeric Cx32 forms are indicated and a β-actin loading control was included for each blot. (E) Immunohistochemical localisation of Cx32 on human bladder and ureter tissues, with rat liver Cx32-positive control tissue. Arrows indicate the focus of Cx32 gap junctions in the basolateral membrane of superficial cells. Scale bar = 25 μm. Source data are available for this figure.

Article Snippet: The PPARγ antagonist T0070907 (T007; Cambridge Bioscience Ltd.) was used at 5 μM by pre-treatment for 3–4 h before induction of differentiation.

Techniques: Gene Expression, In Vitro, RNA sequencing, Biomarker Discovery, Immunofluorescence, Cell Culture, Control, Western Blot, Immunohistochemical staining, Positive Control, Membrane

Journal: Life Science Alliance

Article Title: Connexin 32 constrains a mesenchymal-like switch in differentiated urothelium and luminal cancers

doi: 10.26508/lsa.202503427

Figure Lengend Snippet: (A) RNA was collected from bladder-derived NHU cell cultures, following growth in KSFMc (undiff), 2 mM [Ca 2+ ] for 5 d, or following differentiation in ABSCa (7 d) or TZPD (6 d). GJB1 quantitative RT-PCR was performed with expression normalised to the housekeeping gene GAPDH . Data represent mean fold-change values (±SD) from three technical replicates from a single NHU cell line. (B) NHU cells were pre-treated with a PPARγ antagonist, T0070907 (5 μM) or vehicle control for 3 h, before instigating differentiation using TZPD. RNA was harvested for analysis at 6, 24, 48, and 72 h post-treatment. At each time point, a vehicle control (0.1% DMSO) was included. GJB1 quantitative RT-PCR was performed, with expression normalised to the housekeeping gene GAPDH . Values represent GJB1 expression as fold-change relative to the 6 h vehicle control. Mean fold-change values (±SD) from three technical replicates are shown, from a single NHU cell line. (C) Cx32 immunofluorescence labelling of NHU cells cultured on glass slides for 72 h in TZPD, with or without pre-treatment for 3 h with T0070907 (5 μM). Nuclei are counterstained (blue) with Hoechst 33258. Scale bar = 25 μm. (D) Cx32 immunoblotting of NHU cultures for 48 h of DMSO vehicle control or TZPD. Where indicated, 12.5 μM MG132, a membrane-permeable proteasome inhibitor, was added for the final 24 h before cell lysis. Monomeric and dimeric Cx32 protein products are indicated. β-actin was included as a loading control and claudin 4 immunoblotting was included as an internal MG132 treatment-positive control because claudin 4 is targeted for ubiquitin-degradation in undifferentiated urothelial cells . Source data are available for this figure.

Article Snippet: The PPARγ antagonist T0070907 (T007; Cambridge Bioscience Ltd.) was used at 5 μM by pre-treatment for 3–4 h before induction of differentiation.

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Control, Immunofluorescence, Cell Culture, Western Blot, Membrane, Lysis, Positive Control, Ubiquitin Proteomics